American Herbal Pharmacopoeia

Aloe Vera Leaf

Aloe Vera Leaf Juice
Aloe Vera Inner Leaf Juice
Aloe vera (L.) Burm. f.
Standards of Identity, Analysis, and
Quality Control
Editor
Roy Upton RH DAyu
Associate Editor
Pavel Axentiev MS
Research Associate
Diana Swisher MA
Authors Analytical Josias Hamman PhD James Neal-Kababick
Maltodextrin Assay Tshwane University of Technology Flora Research Laboratories
History Elan Sudberg Pretoria, South Africa Grants Pass, OR
Aviva Romm MD CPM Herbalist Alchemists Laboratories James Harnley PhD Len E Newton PhD FLS
Tufts School of Medicine Costa Mesa, CA United States Department of Kenyatta University
Boston, MA High Performance Thin Layer Agriculture Nairobi, Kenya
Chromatography (HPTLC) Beltsville, MD Eike Reich PhD
Roy Upton RH DAyu
American Herbal Pharmacopoeia® Judy Nichols Ernst van Jaarsveld PhD CAMAG Laboratory
Scotts Valley, CA CAMAG USA South African National Biodiversity Muttenz, Switzerland
Wilmington, NC Institute Tom Reynolds BSC MSc ARCS
Botanical Identification High Performance Liquid Pretoria, South Africa Jodrell Laboratory
Sophie Neale PhD Chromatography (HPLC) Ping Jiao PhD Royal Botanic Gardens, Kew
Royal Botanic Garden Paula N Brown PhD Unigen Inc. London, UK
Edinburgh, UK British Columbia Institute of Seattle, WA
Technology Wenwen Ma PhD
Macroscopic Identification Burnaby, British Columbia, Canada Unigen Inc.
Lynette Casper BA Proton Nuclear Magnetic Resonance Seattle, WA
Planetary Herbals Spectrometry (1H NMR) Michael McGuffin
Scotts Valley, CA John Edwards PhD American Herbal Products
Process NMR Associates Association
Microscopic Identification Danbury, CT Silver Spring, MD
Vaishali Joshi PhD International Status Devon Powell
National Center for Natural International Aloe Science Council
Products Research Josef Brinckmann Silver Spring, MD
University of Mississippi Traditional Medicinals
Sebastopol, CA Santiago Rodriguez PhD
University, MS Lorand Laboratories
Prof Dr Reinhard Länger Houston, TX
AGES PharmMed Reviewers
Vienna, Austria
Ezra Bejar PhD Final Reviewers
Commercial Sources and Herbalife
Los Angeles, CA Kristie M Adams PhD
Handling United States Pharmacopeia
Ken Jones Anna Bozzi Nising Rockville, MD
Aloecorp Inc. Office for Biotechnology, Nutrition
& Consumers at scienceindustries David Cutler PhD
Seattle, WA Jodrell Laboratory
Zurich, Switzerland
Roy Upton RH DAyu Royal Botanic Gardens, Kew
American Herbal Pharmacopoeia® Kim Colson PhD Richmond, Surrey, UK
Scotts Valley, CA Bruker BioSpin Corp
Billerica, MA Harry HS Fong PhD
Department of Medicinal Chemistry
Constituents Stefan Gafner PhD and Pharmacognosy
Jianping Zhao PhD Tom’s of Maine University of Illinois at Chicago
National Center for Natural Kennebunk, ME Chicago, IL
Products Research Ferdinant Malan Du Preez BA (SA) Olwen M Grace PhD
School of Pharmacy, Thad Cochran Capetown, South Africa Jodrell Laboratory
Research Center John S Haller Jr PhD Royal Botanic Gardens, Kew
University, MS Department of History Richmond, Surrey, UK
Qi Jia PhD Southern Illinois University
Unigen Inc. Carbondale, Illinois
Seattle, WA

ISBN: 1-929425-29-5 ISSN: 1538-0297

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data. All efforts have been made to ensure the accuracy Cover Photograph
of the information and findings presented. Those seek-
ing to utilize botanicals as part of a health care program Harvesting Aloe vera in Gonzales,
should do so under the guidance of a qualified health Mexico. Photograph courtesy of
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American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

Ta b l e o f C o n t e n t s
Introduction 2 A l o e Ve r a I n n e r L e a f
A l o e Ve r a L e a f Juice
Nomenclature 2 Nomenclature 43
Botanical Nomenclature Definition
Botanical Family
Definition
Common Names
Identification 43
Macroscopic Identification
Organoleptic Characterization
History 2
Commercial Sources and Handling 43
Identification 7
Botanical Identification
Macroscopic Identification
Constituents 43
Organoleptic Characterization
Microscopic Identification Analytical 43
Maltodextrin Assay
Commercial Sources and Handling 11 High Performance Liquid Chromatography (HPLC)
Sourcing Proton Nuclear Magnetic Resonance Spectrometry (1H NMR)
Sustainability Limit Tests
Cultivation
Harvest References 50
Handling and Processing
Storage
Qualitative Differentiation
Adulterants
Preparations
IASC-Certified Juice Products

Constituents 18

Analytical 22
High Performance Thin Layer Chromatography (HPTLC)
Limit Tests

International Status 27

A l o e Ve r a L e a f J u i c e
Nomenclature 28
Definition

Identification 28
Macroscopic Identification
Organoleptic Characterization

Commercial Sources and Handling 28

Constituents 28

Analytical 29
Maltodextrin Assay
High Performance Liquid Chromatography (HPLC)
Proton Nuclear Magnetic Resonance Spectrometry (1H NMR)
Limit Tests

American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 1

Introduction
The following monographs were developed to establish
guidelines for determining the identity, purity, and quality of
the crude leaves of Aloe vera, as well as aloe vera leaf juice
(made from entire leaf) and aloe vera inner leaf juice (made
from the colorless inner parenchyma) raw materials and
products. These standards are in alignment with the criteria
of the International Aloe Science Council’s certification
program (IASC 2012).
The historical and contemporary uses of Aloe vera
encompass different articles of commerce derived from the
plant. The most widely used medicinal aloe preparation has
been a laxative prepared from the strongly bitter exudate
contained in the aloitic cells of the vascular bundle sheaths
of aloe leaves. The exudate (latex) is typically boiled to a
thickened consistency (inspissated) and dried. This is one of
the oldest continuous drugs in pharmacopoeial history and
is referred to by a variety of names, some of which may cause
confusion with other popular aloe leaf products that have
been introduced over the past few decades. Names applied
to the concentrated and dried aloe exudate include “aloe
latex,” “aloe sap,” “aloe exudate,” “aloe gum,” sometimes
“aloe juice,” and often simply “aloe” or “aloes.” These
preparations are characterized by the presence of phenolic
constituents, particularly aloin A and B (also known as
barbaloin) and chromone aloesin, which are largely
responsible for the laxative effects. Concerns regarding
these compounds as potential carcinogens have recently
been raised (CIR 2007; NTP 2011), resulting in limits
being established by independent organizations (e.g., the
International Aloe Science Council) and restrictions placed
on aloe vera products in some countries (e.g., Argentina,
Bolivia, Brazil). The International Aloe Science Council
(IASC) has developed a set of identity, purity, and quality
standards for aloe vera juice products (i.e., leaf juice and
inner leaf juice) as well as a voluntary certification program.
Products certified by IASC are specifically prepared in a
manner that limits the total amount of aloin A and B in
single-strength raw materials and finished products to 10 ppm
or less, ensures the presence of acetylated polysaccharides at
or above a minimum level (≥ 5% dry weight), and includes
assays to ensure the absence of specific adulterants. Thus,
the pharmaceutical preparations derived from the exudate,
inherently designed for short-term laxative use, must be
clearly distinguished from aloe vera leaf and inner leaf juice
preparations, especially those meeting IASC certification
criteria. Other species of Aloe (e.g., A. ferox) have also
been used in commercial juice products; however, these
species and products are not currently addressed by these
monographs or the IASC certification program.
2 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
Aloe Vera Leaf
Aloe vera (L.) Burm. f.
Nomenclature
Botanical Nomenclature
Aloe vera (L.) Burm. f. syn. A. barbadensis Mill.
Botanical Family
Xanthorrhoeaceae (alternatively placed in Aloaceae and
Asphodelaceae)
Pharmacopoeial Definition
Aloe vera leaf consists of the whole fresh leaves of Aloe vera
(L.) Burm. f., conforming to the methods of identification
provided.
Common Names
English: Aloe vera, aloe, Barbados aloe, burn plant,
Curaçao aloe, lily of the desert, true aloe, West
Indian aloe.
Chinese: Lu hui ye (leaf); lu hui (leaf exudate).
Europe: Aloe vera.
German: Echte Aloe.
French: Aloès, aloès vulgaire.
Portuguese: Babosa, babosa-medicinal, erva-babosa.
Spanish: Acibar, aloe, sávila.
History
The plant genus Aloe has a history of economic and
medicinal use that spans thousands of years and is the
source of some of the oldest known herbal medicines. The
name “aloe” comes from the Greek αλοή (aloí), supposedly
derived, in its turn, from the Hebrew allal or the similar
Arabic word alloeh, both meaning bitter — a tribute to the
taste of the leaf exudate (Park and Lee 2006). The name
aloe is also commonly applied to various products derived
from the plant.
The specific epithet vera in the botanical name Aloe
vera is Latin for truth (hence the common name “true
aloe”) and was first applied to the plant by Linnaeus (1753)
as the name of a variety within a species which he named
Aloe perfoliata. Fifteen years later, two other botanists
independently raised the variety to a full species level: Philip
Miller, in his book The Gardeners Dictionary, referred to
the plant as Aloe barbadensis — the name which may have
been accepted as correct, had not Nicolaas Laurens Burman
named it Aloe vera in his Flora Indica published at least ten
days earlier (Newton 1979; Stafleu and Cowan 1976, 1981).
Publication precedent dictates that the earlier nomenclature
of Burman be accepted (McNeill et al. 2012), and thus the

Figure 1 Early woodcut of Aloe vera
Source: Krauterbuch (Bock 1565).
current nomenclature is ascribed to him and Linnaeus, as
Aloe vera (L.) Burm. f. Nevertheless, many continue to use
the name A. barbadensis Mill.
Early Historical Use
In ancient Egypt, aloe was reportedly depicted in engravings
on a temple dating from as early as 4000 BCE, recorded as
a “sanctuary plant of immortality” (Park and Lee 2006), and
used as a funerary gift to the pharaohs (Ulbricht et al. 2007).
The earliest known record of medicinal application of aloe
dates to a Sumerian clay tablet from approximately 2200
BCE (Park and Lee 2006), indicating the use of the plant
in what is now Iraq over 4000 years ago. There are mixed
records regarding the occurrence of aloe in the ancient
Egyptian medical work Ebers Papyrus (ca. 1534 BCE). A
translation of Ebbell (1937) identifies some form of aloe
as an ingredient in two formulas for the eyes, while the
translation of the same work by Bryan (1930) does not record
any such use. Some historical material suggests that aloe had
become an important medicinal botanical in Greece by the
4th century BCE, and an apocryphal legend arose that when
Alexander the Great conquered the island of Socotra, known
as the source of the highest quality aloe, he replaced the
original inhabitants with Greeks to ensure a steady supply of
the raw material (Flückiger and Hanbury 1879).
Regardless of whether the Greek trade in aloe was
broadly established in the time of Alexander or at a later
date — a suggestion made by Scarborough (1982) — it is
certain that products derived from plants of this genus
were widely used by the beginning of the Common Era
in many of the countries in and around the eastern part
of the Mediterranean Basin. In the first century CE, both
the Greek physician Dioscorides and the Roman natural
philosopher Pliny the Elder extolled the virtues of aloe. In
many cases, there is no possibility to determine the species
these early writers were discussing and whether they were
referring to aloe latex (historically, the most common
article of commerce made from aloe) or other preparations.
While many historical descriptions clearly refer to the latex,
others could be related to the inner leaf. Dioscorides, in
his De Materia Medica written around 65 CE, identified
19 different uses and actions of aloe, including those
as a purgative (for constipation), having the “power of
binding” and “loosening of ye belly,” assuaging “ye itching
of ye eye corners” and “ye headache,” “cleansing of ye
stomach” when drunk with water or milk, being useful for
skin afflictions, boils, and stopping bleeding hemorrhoids
when used internally, for healing of ulcers and wounds
when used externally, and noting that “with wine it stays
ye hair falling off” (Gunther 1959). Pliny in his Naturalis
Historia (ca. 77–79) similarly recorded many internal and
topical uses of aloe. In addition to the widespread use of
the latex as a purgative, Pliny writes: “All eye troubles, it is
agreed, are cured by the aloe, but it is specific for itch and
scaliness of the eyelids; it is also good, applied with honey,
especially with Pontic honey, for marks and bruises; for
diseased tonsils or gums, for all sores in the mouth, and for
spitting of blood…” (Rackham et al. 1949). Pliny further
cites the use of aloe for wounds, hemorrhages, dysentery,
and indigestion. Occasionally, fresh leaf or the juice derived
from it are clearly identified. Pliny, for example, informs
us of one sort of aloe that “groweth … in Asia” of which
“they lay the leaves fresh unto green wounds, for they do …
heale wonderfully, like as their juice also.” Moving several
centuries forward to Arabia, Al-Kindi recorded the use of
“aloe juice” in a formula for abscesses “for which the lancet
is not indicated” (Levey 1966). Al-Kindi also described a
treatment for excessive perspiration, instructing that aloe “is
drunk” for 3 days, while saying that the “sticky substance …
from inside the leaf … is rubbed on to arrest the condition.”
In this case, the juice is clearly distinguished from the
“sticky substance,” which, almost certainly, refers to the leaf
exudate, or latex.
Some early references to aloe may have been to plants in
entirely different genera. For example, “aloe” is mentioned
several times in the Old and New Testaments; however,
most of these references may, in fact, refer to an unrelated
plant — the “tree aloe” (Aquilaria agallocha), from which
an aromatic resin can be derived (Lloyd 1921). However,
some suggest that the aloe referred to by St. John in the
New Testament (John 19: 39-40) as the substance used with
myrrh (Commiphora myrrha) to anoint the body of Christ
was, in actuality, aloe vera (Burnett 1852; Callcott 1842).
Both topical and oral use of Aloe sp. was recorded in
China as early as the Tang Dynasty (618–907 CE), and its
topical use for dermatitis was known there by the 8th century
(Morton 1977). As the plant genus came into common
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 3
Table 1 Historical timeline on the medicinal use of Aloe and Aloe vera

4000 BCE Recorded in Ancient Egypt as a “sanctuary plant of immortality” and used as a funerary gift to the pharaohs.
2200 BCE The earliest documented medicinal use of aloe is found on a Sumerian clay tablet.
1550 BCE The Ebers Papyrus, one of the earliest medical works known, describes the healing benefits of aloe for both
internal and external conditions.
356–323 BCE Alexander the Great reportedly conquers the island of Socotra to secure the trade of Aloe perryi throughout
Asia.
Approximately 51 BCE Referred to as “the plant of Cleopatra” by ancient Egyptians, allegedly regarding its use as a beauty aid.
27 BCE–14 CE Aloe vera is introduced into Greco-Roman medicine during the reign of Augustus.
1st century CE “Aloes” are mentioned in the Holy Bible as the substance used with myrrh to anoint the body of Jesus.
41–68 Dioscorides, in his De Materia Medica, writes the first in-depth report of the pharmacologic actions of aloe.
23–129 Pliny reports on the use of aloe for leprous sores and, when topically applied, to reduce perspiration.
618–907 Aloe vera is used medicinally in China, both topically, for dermatitis, and orally.
9 century
th
Aloe is recorded in Anglo-Saxon “leech books.”
960-1279 The official materia medica of the Song Dynasty describes the use of whole ground leaf for the treatment of
sinusitis, fever, skin conditions, and seizures in children.
14th–16th centuries In Europe, aloe is considered a purgative and a topical treatment of wounds and various skin conditions.
1492 Christopher Columbus, upon setting sail for the New World, writes in his journal, “All is well, aloe is on board.”,
introducing aloe to the Americas.
1650–1742 Aloe vera is first imported to London and included as “Barbados aloe” in the London Pharmacopoeia (1650) and
in the London Dispensatory in 1742.
1650–present Aloe latex (inspissated juice) is included in the majority of pharmacopoeias worldwide.
1753 The botanical name Aloe perfoliata var. vera is created by Linnaeus.
1768 Nicolaas Laurens Burman establishes Aloe vera as a separate species. About ten days later Philip Miller
independently classifies it as A. barbadensis; precedent is given to the earlier publication establishing the
nomenclature as Aloe vera (L.) Burm. f.
1810-1820 A variety of aloe preparations are entered into the Massachusetts and the United States Pharmacopeias.
1851 Edinburgh chemists Smith and Smith extract a cathartic principle from aloe and name it aloin.
1867 The “juice” of A. barbadensis (syn. A. vera) is included in the British Pharmacopoeia.
1912 The first commercial aloe vera farm in the US is started in Florida.
1935 Collins and Collins report on the use of Aloe vera to treat radiation burns, stimulating modern research into the
potential benefits of Aloe vera for a variety of skin conditions.
1959 Aloe is included in the US FDA list of approved food additives.
1975–present Aloe “juice” (exudate) is included in the European Pharmacopoeia.
Present Aloe vera products are approved for therapeutic purposes in Australia, Canada, India, and Korea, among other
countries.

use throughout the world, various purgative preparations
derived from Aloe species were found in Britain (as early as
the 10th century) (Flückiger and Hanbury 1879). Later, the
topical uses of aloe for wounds and various skin conditions
were reported (Park and Lee 2006). Aloe was included in the
London Pharmacopoeia at least as early as 1650 in not less
than 21 official preparations (Felter and Lloyd 1898).
Modern Uses of Aloe Vera Leaf
Contemporary use of aloe vera leaf in folk medicine
practices is broadly documented. Traditionally, the leaf is
“filleted” and the fillet (inner leaf) is used either whole
4 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
or mashed. For internal consumption, it is common in
traditional healing practices worldwide to both maintain
the slimy yellow exudate and to rinse it off with cold water
to reduce the bitterness. This is rarely specified in the
ethnobotanical literature. With the exception of the aloe
latex, use of aloe vera predominantly implies the use of the
inner leaf or its juice, with or without the exudate. Dried
latex and traditional inner leaf preparations that include
exudate are typically used for a short duration, thus limiting
exposure to the laxative and potentially carcinogenic
compounds. Conversely, many of the modern aloe vera
leaf juice products are consumed more regularly, making
the removal of the phenolic compounds desirable, as for
example dictated by IASC. When reviewing the traditional
and scientific literature, care must be taken to denote the
specific preparations being discussed.
Topical Use of Aloe Vera Leaf
Traditionally, the inner leaf is prepared for topical use by
filleting (removing the rind) and mashing the colorless
transparent mass into a liquidy pulp. This is applied to the
skin directly (e.g., to boils and infections), with or without
a gauze. Alternatively, the inner leaf may be exposed by
removing the top leaf rind and spiny edges, while leaving the
back rind intact. These preparations may or may not include
various amounts of the exudate, with the latter sometimes
removed by rinsing in cool water (Upton 2012, personal
communication to AHP, unreferenced).
Popular topical uses of the inner leaf of aloe species,
including aloe vera, today include treatment of abrasions,
burns, cancers (as a poultice), inflammation, psoriasis, skin
irritations and fungal infections, UV-radiation damage; as
an emollient; and as a common cosmetic ingredient (Iwu
1993; WHO 1999). Throughout tropical and subtropical
regions where aloe vera is found, the peeled fresh inner
leaf pulp is applied to inflamed eyes and used for all kinds
of skin inflammations, sores, and burns (Iwu 1993; Morton
1977; Pole 2006). In addition to these, there are numerous
other, less common, topical uses of aloe vera throughout
its areas of distribution, including treating cuts, contusions,
headache, sprains; as a poultice; and as a sunscreen and hair
conditioner (Barcroft and Myskja 2003; Honychurch 1986;
Thomas 1997). In Ayurveda, a traditional medical system in
India, aloe vera is commonly used in sunburn, minor cuts,
insect bites, as a wound healing agent, anti-inflammatory,
and in the treatment of frostbite and psoriasis (Agarwal
and Sharma 2011). Use of fresh aloe vera leaf in India also
extends to eye trouble: the fresh pulp, rinsed in cold water
and mixed with a small amount of burned alum, is wrapped
in muslin fabric and applied to sore eyes (Kirtikar and Basu
2006). In Northwest Mexico, the traditional use of aloe vera
leaf includes topical application of the juice for burns, cuts,
bruises, and rashes (Yetman and Van Devender 2002). In
addition, in Mexico, aloe vera is the most widely used topical
application for the treatment of abscesses in intravenous
drug users (Pollini et al. 2010). In the US Virgin Islands,
apart from the widespread topical use of aloe vera inner leaf
for burns, the leaf pulp is applied as a dressing on wounds to
draw out infection (Upton 2012, personal communication
to AHP, unreferenced). External use of the peeled leaf or
juice is recorded in the Bahamas for treatment of bruises,
boils, carbuncles, sunburn, and cuts (Eldridge 1975). In the
Dominican Republic, in addition to many of the mentioned
uses, aloe vera leaves are applied to the fingers of children
to prevent sucking, rubbed on breasts to encourage weaning,
and rubbed on the body to prevent perspiration, to conceal
the human scent during hunting, and as an insect repellant
(Honychurch 1986; Morton 1977; Thomas 1997).
Clinical interest in the external application of aloe vera
began in the 1930s with a publication of a case report of
a successful treatment of radiation burns with macerated
fresh inner leaf (Collins and Collins 1935). Subsequent
scientific evaluation of the efficacy of the various topical
uses of aloe has produced mixed results. A number of studies
and reviews support the use of external preparations of aloe
vera for the prevention and treatment of radiation burns,
dermatitis, genital herpes, psoriasis, skin inflammation,
sepsis, and wound healing (Akhtar and Hatwar 1996; Bosley
et al. 2003; Chitra et al. 1998a, 1998b; Jia et al. 2008;
Maenthaisong et al. 2007; Moghbel et al. 2007; Ulbricht et
al. 2007; Vogler and Ernst 1999; Yun et al. 2009). However,
some studies report negative effects (e.g., Gallagher and
Gray 2003; Marshall 1990; Richardson et al. 2005; Vogler
and Ernst 1999). Such contradictory findings may stem
from various reasons, from limitations in study designs
and outcome measures to variations in composition of the
preparations used — partially due to lack of quality control
in the manufacturing process (Borrelli and Izzo 2000; Choi
and Chung 2003; Maenthaisong et al. 2007; Vogler and
Ernst 1999). In a published analysis of commercial products
(Bozzi et al. 2007), several products exhibited very low levels
of acetylated polysaccharides (also known as acemannan),
which are recognized as the major bioactive constituents of
aloe vera preparations that do not include the latex.
Internal Uses of Aloe Vera Leaf
Numerous internal uses of aloe vera leaf juice are reported
in India, Pakistan, Africa, the Caribbean, Central and South
America, and the South Pacific. Indications for internal
use include diabetes, coughs and sore throat, kidney pains,
digestive problems, stomach ulcers, and jaundice. The
juice is also used as a mild laxative and for relief of difficult
childbirth. Mixed in rum, the juice of aloe vera leaf is used
as a carminative; with sugar, to relieve asthma and other
bronchial afflictions, and with milk for dysentery in children
(Katewa et al. 2004; Morton 1961, 1977; Qureshi and Bhatti
2008; Thomas 1997; Yetman and Van Devender 2002).
Modern Ayurvedic practitioners use the fresh or powdered
inner leaf of aloe as a general tonic for the circulatory,
digestive, excretory, and female reproductive systems; and
specifically in the treatment of fever, constipation, obesity,
inflammatory skin conditions, lymphadenitis, conjunctivitis,
joint inflammation, jaundice and hepatitis, menstrual
dysregulation, and tumors (Frawley and Lad 1986; Grover
et al. 2002). In the Caribbean, peeled leaf and juice of aloe
vera are consumed, with or without salt, to treat colds, sore
throat, and constipation, and to “keep the blood good”
(Eldridge 1975; Upton 2012, personal communication to
AHP, unreferenced). Aloe vera was reported as the third most
widely used botanical medicine in Jamaica (Picking et al.
2011). Healers from the Dominican Republic report the use
of aloe vera for the treatment of uterine fibroids, menstrual
dysregulation, as an abortifacient, and for “cleansing the
body” (Ososki et al. 2002).
Various preparations of aloe vera leaf have been studied
for a range of internal uses, including ulcers (Feily and
Namazi 2009; Klein and Penneys 1988; Vogler and Ernst
1999; WHO 1999), irritable bowel syndrome (Boudreau
and Beland 2006; Störsrud et al. 2009), atherosclerosis (Patel
and Mengi 2008), hyperlipidemia (Kim et al. 2009), diabetes
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 5
(Grover et al. 2002; Kim et al. 2009), and HIV infection and antifungal activities (Habeeb et al. 2007; Nidiry et al.
(Kahlon et al. 1991; McDaniel and McAnalley 1987). Aloe 2011; Rosca-Casian et al. 2007). Potential mechanisms
vera “gel” (inner leaf juice) was shown in a randomized, associated with the anti-inflammatory effects of aloe vera
double-blind, placebo-controlled trial to be of benefit in include inhibition of thromboxane B2 and prostaglandin
ulcerative colitis (Langmead et al. 2004). In addition, in F2 (Robson et al. 1982), antibradykinin activity (Bautista-
a small double-blind, placebo-controlled, crossover trial, Pérez et al. 2004), and modulation of cyclooxygenase and
freeze-dried aloe vera concentrate reduced the symptoms lipoxygenase (Das et al. 2011; Vázquez et al. 1996).
of interstitial cystitis (Czarapata 1995). Studies on internal
Listings in Official Compendia
use of various “gel” (i.e., inner leaf or its juice) products
While dried aloe latex has been widely recognized as an
suggest anti-inflammatory (Störsrud et al. 2009), antioxidant
official drug throughout the world, other preparations
(Liu et al. 2007; Yu et al. 2009), hepatoprotective (Chandan
from aloe vera are generally not included in international
et al. 2007), hypoglycemic (Bunyapraphatsara et al. 1996;
pharmacopoeias. The World Health Organization developed
Kim et al. 2009; Yongchaiyudha et al. 1996), hypolipidemic
a monograph on aloe vera “gel” (i.e., inner leaf juice) (WHO
(Kim et al. 2009), immunomodulatory (Yu et al. 2009), and
1999), citing numerous references of its use for wound
vulnerary effects (Atiba et al. 2011), as well as promotion of
healing and burn treatment, noting, however, that, as of
increased fat metabolism (Misawa et al. 2008). Preparations
the time of the writing, no uses of the “gel” were supported
derived from the plant and described as “freeze-dried juice
by clinical data. In a monograph developed under the
… heated for 15 minutes at 80 degrees” or “liquid Aloe vera
Canadian regulatory agency Health Canada, external use of
extract” have demonstrated in vitro bactericidal activity
the inner leaf was approved for minor abrasions, burns, cuts,
against a number of pathogenic organisms, including
and for wound healing (NHPD 2006). The inner leaf of aloe
Candida albicans and Streptococcus spp. (Lorenzetti et al.
vera is included as a remedy for burns in the Thai Herbal
1964; Robson et al. 1982). In other studies, aloe vera and its
Fundamental Public Health Drug List (Maenthaisong et al.
constituents have also been shown to possess antibacterial
2007). Preparations of aloe vera, as well as other aloe species

2a. 2b.
Figure 2 Historical illustrations of Aloe spp.
Image sources: (2a) Medicinal Plants (Bentley and Trimen 1880); (2b) Medizinal Pflanzen (Kohler 1890).
Note: Aloe vulgaris Lam. is a former synonym of Aloe vera.

6 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

(e.g., A. ferox and A. perryi), have been accepted for topical
medical applications for management of pressure and stasis
ulcers, post-surgical incisions, and first- and second-degree
burns in the United States.
Concerns Regarding Toxicity
Recently, concerns have been raised regarding the
potential toxicity of topical and orally consumed aloe vera
preparations, due to the high concentration of phenolic
compounds (specifically, aloin A and B) that naturally occur
in aloe vera leaf. The US National Toxicology Program
(NTP) studied the potential carcinogenicity of internal
use of nondecolorized whole leaf extract of aloe vera, with
aloin A concentrations averaging 6300 ppm, in two animal
models, in a long-term (2 years) toxicity study (NTP 2011). A
significant increase in the occurrence of tumors was observed
in the colons of rats, but no such increase was evident in
mice, even with double the maximum dose. The NTP study
did not include leaf preparations made in a manner that
minimizes or eliminates aloin A and B content, nor did they
test inner leaf juice products, which contain relatively low
concentrations of these compounds. These safety concerns
lead IASC to establish limitations on concentrations of
phenolic compounds in aloe vera juice products for oral
consumption. Specifically, IASC certification requires
single-strength raw materials and finished aloe vera products
intended for oral consumption to contain 10 ppm or less
of total aloin. The NTP program also reported that topical
application of aloe vera preparations (including decolorized
and inner leaf juices) resulted in a weak but enhanced
carcinogenic activity of simulated solar light in animals.
This resulted in an expert panel of the Cosmetic Ingredient
Review (CIR 2007) to establish a limit of no more than 50
ppm of aloin in topical products. However, the CIR (2007)
further notes that several clinical studies of preparations
derived from Aloe vera plants demonstrated no phototoxicity.
Other, short-term, safety studies have been conducted on
products with low concentrations of aloins (Williams et al.
2010), as well as on the primary polysaccharide fraction of
the aloe vera inner leaf (e.g., Lynch et al. 2011a, 2011b; Yagi
et al. 2009), with no serious adverse events reported.
The uncertainties about safety and the ambivalent
findings of research studies have not dissuaded the general
public from using aloe vera products as a primary treatment
for sunburn, or manufacturers from including it as a key
ingredient in balms, cosmetics, and toiletries, as well
as in foods and dietary supplements. Multiple potential
benefits of internal use of aloe vera leaf products that have
undergone appropriate processing in accordance with the
modern knowledge of the chemistry of the plant have also
been identified. Aloe vera provides us with rich, ancient, and
multicultural history and medical legacy. In the meantime,
the plant continues to be used throughout the world in
thousands of products today, much as it has been for
millennia.
Identification
Botanical Identification
Herbaceous perennial, succulent, erect, suckering freely to
form large colonies. Stem: Short, up to 30 cm, or absent.
Leaves: Spirally arranged as a clustered rosette, fleshy,
yellowish-green to glaucous (young plants may have white
spots), lanceolate, 40–60 x 6–7 cm, with hardened pale
teeth, 2 mm long, 10–20 mm apart, along the margin.
Inflorescence: Erect, to 1 m, with 1–3 branches; raceme
10–40 cm. Flowers: Yellow to red*, perianth in 2 whorls, the
outer whorl fused for less than half of its length, cylindric or
slightly swollen below, glabrous, 20–40 mm; pedicels stout,
approximately 6 mm long, subtended by bracts; bracts 10
mm; stamens 6; ovary superior, 3-celled, with many ovules
in each cell; anthers and styles slightly projecting from the
perianth. Fruit: A woody capsule with many black seeds
(Carter et al. 2011; Holmes and White 2002; Long and
Lakela 1971; Wood 1997).
*
The flower color of Aloe vera is reported as yellow in some botanical
literature but wild specimens can have both red and yellow flowers, as do
many other aloes (Wood 1997). Different color forms of Aloe vera available
commercially may therefore be due to this genetic heritage or the result of
hybridization with other species or cultivars.
Distribution: Found growing in stony outcrops and sandy
plains, roadsides, and similar places, in full sun; sea level to
1300 m. Flowers in winter and spring, occasionally in other
seasons. Aloe vera is cultivated widely in southern Texas and
is naturalized in Arizona, Florida, Texas, and many tropical
and subtropical countries. The exact origin of the species
is unknown but is likely to be southwest of the Arabian
peninsula, where the nearest relative, Aloe officinalis Forssk.,
is still growing wild (Carter et al. 2011; Holmes and White
2002; Long and Lakela 1971; Wood 1997).
Macroscopic Identification
The leaf of aloe vera can be described as consisting of two
major parts: the outer green rind and the colorless inner
leaf. The inner leaf, alternatively referred to as “gel,” “pulp,”
“mucilage layer,” “aquiferous tissue,” or “mesophyll,” is a
clear, transparent, colorless mass, taking most of the leaf’s
diameter, which consists of parenchymatous cells that
contain clear liquid constituting what is known as aloe vera
inner leaf juice (also see the accompanying Aloe Vera Inner
Leaf Juice monograph). Between the rind and the inner leaf,
a vascular layer can be distinguished, containing a series of
tubules (vascular bundles), which run along the full length
of the leaf.
Fresh Leaf
Surface view: Blade thick, succulent, bayonet-shaped,
narrowly-lanceolate, 30–50 cm in length, 10 cm wide at
base, long-acuminate; color green to glaucous, with whitish
spots in younger plants; margin has pale, white to redddish
or light-brown teeth or spines (Bailey and Bailey 1976;
Culbreth 1917; WHO 1999). In harsher growing areas with
limited water supply, the leaves tend to grow thicker and
have more parenchymatous tissue.
Transverse section: Adaxial surface slightly concave; abaxial
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 7
3a. 3b. 3c.

3d. 3e. 3f.

Figure 3 Botanical characteristics of Aloe vera

3a. Cultivated A. vera.
3b. Naturalized A. vera growing wild in Brazil.
3c. Aloe vera inflorescences.
3d. Aloe vera flowers.
3e, 3f. Cultivated A. vera.
Photographs courtesy of: (3a) Aloecorp, Inc, Lacey, WA; (3b) Len Newton;
(3c-f) Steven Foster Photography, Fayetteville, AR.

8 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

4a. 4b.
Figure 4 Other medicinal species of Aloe
4a. Flowering A. ferox.
4b. Flowering A. arborescens.
Photographs courtesy of Ernst van Jaarsveld, the South African National Biodiversity Institute, Pretoria, South Africa.

surface markedly convex. The following layers or zones can
be distinguished: outer epidermal layer with thick cuticle;
green chlorenchyma layer; brown or reddish-brown ring of
vascular bundles from the parenchymatous sheath of which
the bitter, yellow sap exudes; and central parenchyma tissue
appearing as a mucilaginous clear “gel” and occupying
much of the leaf’s diameter (Evans 2002; Longo 2002; Panda
2004; Surjushe et al. 2008). The cuticle, the epidermis,
and the chlorenchyma constitute the rind. Some sources
(e.g., Barcroft and Myskja 2003) further separate the inner
parenchyma into “mucilage” and “gel” layers.
Species Differentiation
Several other species of Aloe are used globally, including
for manufacturing of juice products. The distinguishing
morphological characteristics of Aloe vera compared to
some other common Aloe species are provided in Table 2.
Occasionally, other, less common species, mainly occurring
in continental Africa and Madagascar, enter the juice
market (Grace 2011).
Organoleptic Characterization (inner leaf)
Taste: Mild, bland.
Aroma: Faint, characteristic.
Texture: Very slimy, tacky.
Microscopic Identification
The leaf of aloe vera consists of the following tissues:
epidermis, chlorenchyma, vascular bundles, and colorless
inner parenchyma. The microscopic anatomy of Aloe leaves
is fairly constant regardless of the species. Some species may
be distinguished by microscopic analysis of the leaf surface
(Cutler 2004; Li et al. 2003).
Epidermis
In transverse section, the leaf epidermis on both the adaxial
and abaxial surfaces consists of a single, uniform layer of
cells, approximately 25–40 µm thick, covered with a waxy
thick cuticle, approximately 6–8 µm. The epidermal cells
are arranged in parallel with the long axis of the leaves
and contain few chloroplasts. Both surfaces of epidermis
have sunken stomata, with guard cells surrounded by 4–5
epidermal cells.
Chlorenchyma
Just below the epidermis, there are usually 8–10 layers
(the number of layers can vary) of hexagonal-to-rounded
chloroplast-containing chlorenchyma cells, approximately
50–60 µm wide. Some idioblasts, with needle-shaped oxalate
crystals, are also present. Chlorenchyma in Aloe species is
not differentiated into spongy and palisade layers.
(continued page 11)

Table 2 Macroscopic characteristics of Aloe vera leaf compared to leaves of other Aloe species occurring in trade worldwide

Aloe vera A. ferox A. arborescens A. perryi

Leaf size and form 40-60 cm long, 10 cm Up to 100 cm long, 15 cm 50-60 cm long, 5-7 cm wide, Up to 35 cm long,
broad at base broad at base sickle-shaped, frequently approximately 7.5 cm
recurved wide

“Teeth” (color, size, and Pale teeth, 2 mm long, Dark brown, both along 3-5 mm long, along the Pale brown, ~4 mm long,
location) 10-20 mm apart the margins and on the margins, 5-20 mm apart along the margins, 6 mm
upper and/or lower leaf apart
surfaces
Based on Carter et al. 2011; van Wyk and Smith 2003.

American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 9

5a.

5b. 5c.

5d. 5e.

Figure 5 Macroscopic characteristics of Aloe vera leaf

5a. Commercially cultivated Aloe vera leaves.
5b. Aloe vera leaf (upper surface).
5c. Aloe vera leaf (lower surface).
5d. Aloe vera leaf (cut base).
5e. Aloe vera inner leaf fillet.

10 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

6a.
Figure 6 Macroscopic characteristics of other Aloe species
6a. Aloe ferox leaf.
6b. Leaves of Aloe arborescens.
Vascular Layer
A single row of vascular bundles occurs between the
chlorenchyma and the colorless inner parenchyma, which
it encircles. Well-developed parenchymatous cells surround
the vascular bundles, forming a bundle sheath. Sieve tubes
and companion cells are narrow. The phloem is not very
distinct, and the xylem is composed of narrow vessels and
tracheids with annular and spiral thickenings. The yellow
exudate is secreted by clusters of thin-walled “aloitic” cells
forming a cap at the outer (phloem) pole of the vascular
bundles, as part of the bundle sheath.
Inner Parenchyma
Occupying the center of the leaf, the inner parenchyma
is composed of large parenchymatous cells, approximately
400-500 µm in diameter, that lack chloroplasts and are filled
with polysaccharide-rich juice.
Commercial Sources
and Handling
There are numerous quality control issues associated with
aloe vera products, including differentiating between
closely related species, procuring appropriate raw material,
processing the leaves to reduce the content of the phenolic
compounds, preserving active constituents during processing
and storage, and identifying adulterants. Multiple procedures
have been developed to address these issues. For a detailed
presentation of aloe vera processing see He et al. (2005),
Ramachandra and Rao (2008), and Waller et al. (2004).
Sourcing
The preponderance of aloe vera is grown in Mexico,
followed by southern Texas and Florida in the United States.
Other sources include Argentina, Central America, China,
the Dominican Republic, India, North Africa, Thailand,
and Venezuela.
6b.
Sustainability
The international trade of all Aloe species, except for Aloe
vera, is governed by the Convention on the International
Trade of Endangered Species (CITES 2012), which had
Aloe spp. added to its Appendix II in 1975. Aloe vera has
been specifically excluded from the CITES-listed species
database in February 1995 — likely because the totality of
traded materials from this species comes from cultivated
sources (Grace et al. 2008).
Cultivation
The natural habitats of Aloe vera are almost certainly
subtropical, and the plant grows best when supplied with an
excess of 50 cm of rain annually, in nitrogen-rich, alkaline
soil (Waller et al. 2004). The Rio Grand Valley of Texas
has been identified as an ideal growing region for aloe
vera. Because of its thick and shallow root system, aloe vera
requires well-drained soil and does not tolerate deep tillage
(Waller et al. 2004). Good drainage also helps to prevent
root rot, to which aloe vera is prone. While most species of
Aloe typically grow in sandy soils, aloe vera has also been
shown to grow well in tuff or basalt soils, with well-drained
substrates allowing for better growth (Saks and Ish-shalom-
Gordon 1995). It can also tolerate saline soils, but the
salinity negatively affects biomass (Tawfik et al. 2001).
Aloe vera is reproduced asexually or by seed. Numerous
shoots (pups), developing around the base of mature plants,
may be separated and transplanted when 15–20 cm (6–8
inches) high and will have fully mature leaves in 3 years.
The shoots should be removed from mother plants at
least twice annually to encourage larger leaf growth in the
parent plants. In vitro propagation has been reported to
be an effective way of reproduction, sufficient to meet the
demands of the growing aloe vera industry (Hashemabadi
and Kaviani 2008; Meyer and van Staden 1991).
Planting densities reportedly range 4500–6000 plants
per acre. Morton (1977) reports that to reach maximum
development, with a single mature leaf weighing 1 pound,
low content of latex, and plentiful pulp, aloe vera plants
should be grown in partial shade, irrigated in dry seasons,
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 11
7a. 7b. 7c.

7d. 7e. 7f.

7g. 7h. 7i.

7j. 7k. 7l.

12 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

Figure 7 Microscopic characteristics of Aloe vera leaf
7a. Transverse section of the leaf under stereo microscope.
7b. Transverse section of the leaf under stereo microscope; note the vascular bundles
secreting the exudate, surrounding aquiferous tissue. Exudate was stained red with
potassium hydroxide solution.
7c. Outer part of leaf, showing thick cuticle, epidermis, and chlorenchyma bordering the
aquiferous tissue (20x).
7d. Transverse section, showing epidermal and chlorenchymatous layers. The blackened
matter is the aloe exudate from which aloe latex is derived (20x).
7e. Transverse section, showing epidermis (left), chlorenchyma, vascular bundles with large
surrounding cells, aquiferous tissue (right) (10x).
7f. Transverse section of epidermis with a sunken stoma (40x).
7g. Chlorenchymatous and aquiferous layers.
7h. Vascular and aquiferous layers, showing parenchyma, phloem, and xylem (20x).
7i. Aquiferous layer, showing oxalate acid crystals (20x).
7j. Transverse section, showing idioblast containing raphides (40x).
7k. Raphides separated from tissue (polarized light, compensator first order).
7l. Transverse section showing epidermis, chlorenchyma, and vascular bundle (UV 330-380
nm) (10x).
Microscopic images courtesy of Vaishali Joshi, National Center for Natural Products Research, University
of Mississippi; and Reinhard Länger, AGES PharmMed, Vienna, Austria.

wearing protective clothing, to prevent injury from handling

and well fertilized. Diaz and Gonzalez (1986) also observed
that aloe vera grown under natural shading developed more
biomass than plants grown in full sun. Conversely, one study
showed that plants grown under full sunlight produced
more numerous and larger axillary shoots, resulting in twice
the total dry mass than plants grown under partial shade
(Paez et al. 2000). Sunlight appeared to have no effect on
aloin content or primary and secondary metabolites (Paez
et al. 2000).
Mustafa (1995) reported that a high biomass of aloe
vera was obtained with short (8-day) intervals of irrigation.
In a study on aloe vera cultivated in Chile, Silva et al. (2010)
determined that the highest biomass was produced when the
plants received via irrigation 15% of the mean evaporative
demand of the previous year. The same authors found that
excessive watering (20% of the mean evaporative demand)
resulted in smaller root growth, root rot, and discoloration
of the leaves.
Soil nitrogen should ideally be maintained at
0.40%–0.50%. According to recommendations of IASC,
approximately 50 kg of nitrogen per hectare should be added
to the soil after each quarterly harvest prior to planting and
throughout the year.
Though aloes are generally drought-tolerant plants,
prolonged periods of drought (e.g., 3 years) can decimate
young populations. The plants can withstand short periods
of frost, but the leaves redden and become damaged with
decreasing temperatures. This damage, however, mostly
occurs in the upper part of the leaf and usually results in
negligible loss of juice, unless it progresses to tip necrosis
(see Harvest).
Harvest
Aloe vera leaves are generally ready for harvest after 3
years of age. Proper harvesting is a labor-intensive process.
Collection must be done with heavy gloves and while
the spiny leaves. Typically, the outermost 3–4 leaves are
harvested by pulling each leaf away from the plant stalk and
cutting at the white base. The leaves should be handled
gently. Care should be taken to prevent damage to the outer
rind and to maintain the seal at the base of the leaf in order
to prevent introduction of bacteria. Leaves that show signs of
tip necrosis should not be harvested, as these provide entry
points for microbial contamination. Harvested leaves are
carefully stacked and then transferred to a refrigeration or
processing facility.
According to Wang (2007), at least 15 and, ideally, 18
leaves should be left on the plant to maintain high leaf
yields. It is estimated that aloe leaves can be harvested 3-5
times annually, with individual plants yielding approximately
22–24 leaves or 10–12 kg per year.
Handling and Processing
Aloe vera leaves are typically subjected to a series of
processing techniques. The following paragraphs focus
on describing modern production of aloe vera leaf juices,
excluding the production of aloe latex.
For manufacture of aloe vera leaf juices, processing
should take place as soon as possible due to the highly
perishable nature of the juice, ideally within 36 hours of
harvesting the leaves (Ramachandra and Rao 2008). If
immediate processing is not possible, leaves should be stored
in a refrigerated facility. Prolonged storage of the leaves after
harvest without refrigeration may result in enzymatic and
bacterial degradation of the polysaccharides.
Due to concerns over potential carcinogenicity of aloins
A and B, filtration (“decolorization”), often with activated
charcoal and diatomaceous earth, has become a common
practice in the manufacturing of aloe vera leaf juices.
Decolorization removes a variety of small-molecular-weight
organic compounds, including aloins A and B, from aloe
vera leaf material.
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 13
Figure 8 Harvesting Aloe vera leaves (Gonzales, Mexico)
Photograph courtesy of Aloecorp Inc., Lacey, WA.
aqueous formulations.
Following enzymatic treatment, the material is subjected
Crude Aloe Vera Juice
to a series of filtration steps to remove any remaining
Traditional cultures worldwide utilize aloe vera juice by
rind particles and the undesired phenolic compounds.
expressing the juice from the freshly filleted leaves or by
Various modifications and proprietary variations of the
mashing the inner leaf and consuming the resulting pulp
filtration process exist. The material is then passed through
as is or mixed with water. This crude juice can be prepared
a depulping machine, similar to that used for depulping of
with or without the exudate. The exudate can be removed
citrus fruits. This is immediately followed by sterilization
by rinsing the intact gel layer in cool or cold water. These
(e.g., pasteurization), which is necessary to prevent
types of preparations have highly variable concentrations of
degradation of the acetylated polysaccharides by various
phenolic compounds, such as aloins A and B.
exogenous and endogenous microorganisms (Waller et
Aloe Vera Leaf Juice al. 2004). Further, to remove aloins A and B and other
Aloe vera leaf juice is prepared from the entire leaf. The phenolic compounds, decolorization with activated carbon
base and tip of the leaf are first removed and the remaining is employed. The resulting product is a clear fluid, which
portion is cut into sections and ground into slurry. In some is similar in organoleptic characteristics to inner leaf juice.
cases the soupy mass is then treated with the enzyme
Aloe Vera Inner Leaf Juice
cellulase, which hydrolyzes polysaccharides, to obtain a
To obtain this type of product, the outer part of the leaf (“the
more liquid product. However, since cellulase hydrolyzes
rind”) is separated from the clear inner leaf and discarded
the same β-(1→4) glycosidic bonds that occur in aloe vera
prior to expressing the juice. This process, when properly
acetylated polysaccharides, excessive enzymatic treatment
done, can minimize the presence of phenolic compounds
(overprocessing) may result in a significant loss of these
in the finished juice product.
compounds, which are of primary medicinal interest in
In modern production, the leaves are initially subjected
the juice. For example, Waller et al. (2004) reported
to washing with an antimicrobial agent (e.g., hypochlorite)
50-90% breakdown of glucomannans in aloe vera leaf juice
to remove dirt and surface bacteria. Often the exudate is
produced using the enzymatic treatment. Inactivation of
then allowed to drain from the bottom portion of the leaf
endogenous or exogenous cellulase (e.g., by denaturation)
(He et al. 2005). This is followed by culling, trimming, and
is necessary in order to stabilize the polysaccharides in
14 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
Figure 9 Mechanical filleting and depulping of Aloe vera leaf
Photographs courtesy of Aloecorp Inc., Lacey, WA
removing the rind to yield the leaf “fillet” in the process
called “filleting.” Filleting can be done either by hand
or mechanically. According to some researchers (e.g.,
Ramachandra and Rao 2008; Waller et al. 2004), hand
filleting is the best way to ensure absence of the rind and
latex. With hand filleting, the lower inch of the base of
the leaf, the leaf tip, spines along the leaf margin, and top
and bottom rinds are removed manually. For mechanical
filleting, several machine types are used. In one of them,
the leaf is placed on a conveyor belt and passes through
a set of knives, while rollers firmly press against the rind,
expressing the inner leaf. If the tension of the rollers is too
high, excessive concentrations of the phenolic compounds,
which reside in the vascular layer, can result; if the roller
tension is too low, some inner leaf material will be discarded
and wasted. In other types of machines, the leaf may be fed
through the filleter by hand. To further reduce the chance of
contamination of the inner leaf material with the phenolic
compounds of the exudate, the inner leaf “fillets” are then
rinsed in flowing water (He et al. 2005).
After filleting, the inner leaf material is subjected to
depulping, usually followed by pasteurization. Subsequent
processing may include enzymatic treatment, filtration,
dearation, decolorization by activated carbon, and addition
of preservatives. During all processing stages, adherence to
proper sanitary procedures is critical, as aloe pulp and juice
provide a rich medium for the growth of bacteria, which
may affect the chemical composition of the material and the
characteristics of the finished product.
Pasteurization
Pasteurization is typically performed in production of both
types of aloe vera leaf juices as soon as the liquid material
is obtained, in order to reduce bacterial degradation of the
key juice constituents (i.e., acetylated polysaccharides),
which can happen very rapidly. However, pasteurization,
particularly if prolonged, can also have a detrimental effect
on the structure of the polysaccharides, which are also prone
to thermal degradation.
Dry Concentrates
Aloe vera leaf juices are frequently marketed as dried
concentrates, either as powder or flakes (also see
“Preparations”). Drying aloe vera juices begins with
concentrating the juice to a higher level of solids by utilizing
evaporators of various kinds, which increase solids content
of the juice from as low as 0.46% to a range of 10%–20%.
Subsequent drying methods include spray-drying, freeze-
drying, and belt-drying. The resulting products are typically
sized at 10–80 mesh.
Drying at a temperature of 60 ºC reportedly resulted in
only minor alterations in physiochemical properties of an
inner leaf product (Miranda et al. 2009). Drying at higher
temperatures resulted in changes in the average molecular
weight of acetylated polysaccharides from 45 kDa to 75 and
81 kDa in samples dried at 70 ºC and 80 ºC, respectively
(Femenia et al. 2003). However, Chang et al. (2006)
reported that drying at 70 ºC resulted in the maximum
preservation of polysaccharides, with total concentrations
decreasing—at temperatures below 70 ºC due to the activity
of the natural enzymes present in aloe vera, and at higher
temperatures due to thermal degradation.
Storage and Stability
Whole aloe vera leaves can begin degrading within six hours
after harvest (Ramachandra and Rao 2008). The juices,
if not preserved, degrade over a relatively short period of
time due to enzymatic and microbial activity and oxidation.
Dried juice concentrates are more stable; however, they
are also prone to degradation if exposed to humidity and
heat. For these reasons, stabilizing agents and preservatives
are typically added to aloe vera leaf juices during the
manufacturing process. Sodium sulfite or sodium benzoate
are used to prevent microbial growth. Sorbate, citrate, or
ascorbate are added to prevent oxidation. Citric acid is
commonly used to adjust pH to < 4.6. In one study of aloe
vera inner leaf products, the addition of 1% citric acid and
1000 ppm of sodium benzoate was optimal for stabilizing
the juice (Hemalatha et al. 2008).
Liquid products should be refrigerated after opening
and protected from agitation, as aloe vera leaf juices are
sensitive to oxidation.
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 15
Qualitative Differentiation
When assessing quality of unprocessed aloe vera leaf
material, special attention should be paid to the structural
integrity of the leaves, as any damaged tissue (e.g., tip
necrosis), even in a minor portion of the leaves, can become
a site for microbial contamination, which will potentially
affect the quality of all later stage material.
A distinct characteristic of unprocessed aloe vera leaves
is their content of phenolic compounds (e.g., anthrones,
chromones, and anthraquinones). These constituents occur
exclusively in the vascular layer of the leaves and are absent
in the clear inner leaf. With safety concerns regarding these
compounds, the aloe vera leaf juice industry began using
activated charcoal in the process called “decolorization.”
This practice can consistently reduce the concentration
of phenolic compounds in finished aloe vera leaf juice
products. In the case of manual or mechanical separation of
the inner leaf, simply washing the inner leaf fillets prior to
further processing removes most of the phenolics. However,
due to inadequate techniques used, these compounds may
still occur in non-decolorized inner leaf juice products. In
IASC-certified products intended for oral consumption, the
total content of aloins A and B should be less than 10 ppm
(based on single strength of the raw material or finished
product). In aloe vera leaf juice products to be utilized
topically in cosmetic products, the limit is set as less than 50
ppm (CIR 2007).
Qualitative parameters that can be applied to aloe vera
leaf products include content of compounds of medicinal
interest. In this regard, acetylated polysaccharides have
been extensively researched and are considered some of
the most biologically active components in aloe vera leaf.
Currently, the only validated method for determination
of the content of this group of compounds in materials to
be used in consumer products is proton nuclear magnetic
resonance spectrometry (1H NMR), described in the
Analytical section of the Aloe Vera Leaf Juice monograph.
Certain processing procedures involved in manufacturing
of aloe vera leaf products—particularly, pasteurization and
cellulase treatment—directly affect polysaccharide content.
A review of a limited number of commercial aloe vera leaf
products suggests that polysaccharide concentrations vary
widely, with some products containing as low as 1% dry
weight of acetylated polysaccharides (Bozzi et al. 2007). The
term “overprocessed” typically refers to materials that have
undergone prolonged pasteurization and/or uncontrolled
enzymatic treatment and demonstrate almost complete
lack of polysaccharides. Overprocessed juices can also be
distinguished by yellow discoloration due to caramelization
of sugars (even in decolorized material), changes in aroma,
and increased bitterness (Waller et al. 2004). Changes in
color typically occur with aloe juice preparations over time
and can be an indicator of relative freshness. As reported by
Chang et al. (2006), the juice color changes from whitish,
when fresh, to yellow to brown when exposed to high
temperatures or oxidation (see Figure 10c-g).
Several other small molecules can be used as additional
markers of quality of aloe vera leaf products. Acetic acid is
16 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
indicative of the degradation of acetylated polysaccharides.
Lactic acid levels of more than 10% are indicators of
bacterial activity. The presence of succinic and fumaric
acids indicates enzymatic degradation of the polysaccharides
caused by the enzymes naturally present in aloe vera leaf.
If exposed to excessive or prolonged heat, formic acid is
produced due to the thermal degradation of glucose. If juice
products are not processed or stored properly, ethanol is
formed by fermentation processes from wild yeasts. Pyruvic
acid is created from the degradation of carbohydrates.
Detection of these compounds is achievable with the
1
H-NMR method described in the Analytical section of the
Aloe Vera Leaf Juice monograph.
Adulterants
A variety of other Aloe species are traded commercially and
sometimes are not accurately declared as to proper species.
Products misrepresenting the species used are violative of
federal labeling laws. A number of Aloe species (e.g., A.
ferox, A. arborescens, and A. perryi) are readily differentiated
by the shape of the leaves (see “Macroscopic Identification”
and Table 2).
Maltodextrin is commonly used as a carrier during
spray-drying of aloe vera leaf juices in the manufacture
of powdered concentrates. For this purpose, the ratio of
maltodextrin to juice powder is typically 1:1, corresponding
to the concentration of 50% dry weight in the finished
product. Maltodextrin may also be added to artificially
enhance polysaccharide content and has historically been
one of the most common adulterants in aloe vera inner leaf
juice products. If not fully disclosed in labeling, maltodextrin
is considered an adulterant. For methods of detection and
quantitation of maltodextrin, see “Maltodextrin Assay”
and “Quantitative Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR),” respectively, in the Analytical
section of the Aloe Vera Leaf Juice monograph.
Dilution is another issue of concern, as products
may contain added water that can decrease the efficacy
of the product. If undeclared, such products are out of
compliance with labeling regulations. IASC establishes
specific standards of product quality and potency.
Products labeled “aloe vera inner leaf juice” should
consist solely of the liquid from the inner leaf. Isocitrate
(isocitric acid) was established by IASC as a negative marker
for the inner leaf. According to IASC guidelines, aloe vera
leaf juice products that contain more than 5% dry weight
of isocitric acid should be labeled as “aloe vera leaf juice,”
not “aloe vera inner leaf juice” (see “IASC-Certified Juice
Products” below). For estimation of isocitrate content, see
the “Quantitative Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR)” in the Analytical section of the
Aloe Vera Inner Leaf Juice monograph.
Preparations
For the traditional preparation of the inner leaf, the tip,
butt, and toothed margins of the leaf are cut off, and the
inner leaf fillet is separated from the green rind. The latter
is discarded. If the bitter principles of the exudate are
desired, the entire inner leaf can be mashed into a slurry,
cut into cubes, scooped with a spoon, or mixed in a food
processor, with or without added water or juice. To reduce
or eliminate the bitter exudate, the filleted gel layer can be
rinsed in cool water. The fillet can then be mashed, cubed,
or slurried as described above. The fresh juice should be
used immediately.
Commercial preparations from aloe vera leaf include
aloe vera leaf juice (sometimes referred to as decolorized,
purified, or filtered aloe vera whole leaf or whole leaf
extract), aloe vera inner leaf juice (sometimes also referred
to as “aloe vera gel”), and drug aloe (aloe latex). Today,
among the most popular aloe vera preparations are a variety
of liquid and dried aloe vera leaf and inner leaf juice
products of different “strengths” (e.g., single-strength, or 1×;

10a. 10b. 10c.

10d. 10e. 10f.

10g. 10h. 10i.

Figure 10 Preparations of Aloe vera
10a. Aloe vera leaf fillet with rind fragment.
10b. Fresh Aloe vera leaf exudate.
10c. 1:1 non-decolorized Aloe vera inner leaf juice (fresh sample on left; oxidized sample on
right).
10d. 1:1 IASC-certified decolorized Aloe vera inner leaf juice (fresh sample on left; oxidized
sample on right).
10e. 10:1 IASC-certified decolorized Aloe vera inner leaf juice (fresh sample on left; oxidized
sample on right).
10j.
10f. 10:1 non-decolorized Aloe vera inner leaf juice (fresh sample on left; oxidized sample on
right).
10g. 1:1 IASC-certified decolorized Aloe vera leaf juice (fresh sample on left; oxidized sample
on right).
10h. Dry Aloe vera inner leaf juice (flakes).
10i. Dry Aloe vera inner leaf juice (powder).
10j. Dry exudate (inspissated juice, or latex) of A. ferox.
Photographs courtesy of: (10a, c-g) Aloecorp Inc., Lacey, WA; (10b) © 2010 Steven Foster photography,
Fayetteville, AR; (10h-j) American Herbal Pharmacopoeia®, Scotts Valley, CA.

American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 17

double-strength; 5×; etc.). Dry powdered concentrates are
obtained by belt-drying, spray-drying, or freeze-drying. The
convention used in the aloe vera industry is that the typical
solids content of aloe vera leaf juice is 1% weight, while that
of inner leaf juice is 0.5% weight. This approximation is
used to denote the strength of dry powdered concentrates,
e.g., “100× aloe vera leaf juice” is applied to labeling pure
powdered concentrate of aloe vera leaf juice (i.e., aloe vera
whole leaf extract), “200×” is applied to pure powdered
concentrate of aloe vera inner leaf juice, while “100×
aloe vera inner leaf juice” implies that the content of aloe
vera inner leaf material in the product is 50%, with the
remaining part being, typically, maltodextrin (used as a
carrier in spray-drying process). The powders may be sold as
bulk or in capsules. Aloe vera leaf juices are also included as
ingredients in a variety of cosmetic products, or mixed with
thickening agents (e.g., carrageenan) and sold as “aloe vera
gel,” popularly used as a topical application for sunburns
and general skin health, as well as in a variety of household
products (e.g., hand towels, etc.).
Commercial aloe vera leaf products may be standardized
to specific markers or comply with certification standards
(e.g., those of IASC). Finished products typically contain
artificial or natural preservatives (e.g., benzoic acid, sodium
sulfite, and potassium sorbate), pH buffering agents (e.g.,
ascorbic or citric acid), and carriers (e.g., maltodextrin).
Such products are often referred to as “stabilized.” Glucose,
glycerin, and malic acid have also been reported as additives
(Bozzi et al. 2007).
IASC-Certified Juice Products
The International Aloe Science Council (IASC) has
established several qualitative and quantitative parameters
for assessing quality of aloe vera leaf juices (Table 3).
The content of acetylated polysaccharides is considered
a marker of quality with a minimum standard of 5% dry
weight for both IASC-certified aloe vera leaf juice and
aloe vera inner leaf juice raw materials. Due to concerns
over the cathartic and potentially carcinogenic activity of
Figure 11 Certification seal of the International Aloe Science
Council
18 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
Table 3 IASC certification requirements for aloe vera leaf juice
and aloe vera inner leaf juice
Compound Certification requirement
Acetylated mannans ≥ 5% by dry weight
Glucose Present
≤ 10 ppm in single-strength juice,
analysed by HPLC or other fit-for-
Aloin
purpose methodology approved by
the IASC
Must be listed on label and analysis
Maltodextrin
must meet label claims.
≥ 1.0% in single-strength leaf juice;
Solids ≥ 0.5% in single-strength inner leaf
juice
Ash ≤ 40%
Isocitrate ≤ 5% in aloe vera inner leaf juice
aloins A and B, IASC recommends that aloe vera products
and raw materials intended for oral consumption contain
not more than 10 ppm of aloins A and B in single-strength
preparations. The high-performance liquid chromatography
(HPLC) procedure for quantitation of the aloin content in
aloe vera leaf juice and aloe vera inner leaf juice products
can be found in the Analytical section of the Aloe Vera
Leaf Juice monograph. Further, to prevent misbranding
of aloe vera leaf juice as aloe vera inner leaf juice, IASC
has identified isocitrate levels of more than 5% dry weight
as a unique marker of the outer parts of aloe vera leaf
and has included testing for isocitrate content as part of
its certification program for aloe vera inner leaf juice raw
materials (see “Quantitative Proton Nuclear Magnetic
Resonance Spectrometry” in the Analytical section of the
Aloe Vera Inner Leaf Juice monograph).
Constituents
Aloe vera leaves contain a diverse array of compounds,
including anthraquinones (e.g., aloe-emodin), anthrones
and their glycosides (e.g., 10-(1,5’-anhydroglucosyl)-aloe-
emodin-9-anthrone, also known as aloin A and B), chromones,
carbohydrates, proteins, glycoproteins, amino acids, organic
acids, lipids, and minerals (see Table 4). The profile of these
constituents varies greatly depending on the part of the leaf
and the type of processing the material undergoes. The
bitter, yellow-colored exudate, which appears after the leaf
is cut, contains approximately 80 phenolic constituents, the
most abundant being aloins A and B. The inner parenchyma
tissue of aloe vera leaves contains a large amount of water
(ca. 98.5%), and the remaining solid material consists of
carbohydrates, organic acids, and small amounts of other
compounds, such as proteins, vitamins, lipids, and amino
acids (primarily arginine, asparagine, serine, aspartic acid,
Table 4 Compounds identified in Aloe vera leaf

Polysaccharides Anthrones and Fatty Acids Vitamins Amino Acids Minerals

anthraquinones*
Acetylated Aloe-emodin Capric acid b-Carotene Arginine Potassium
glucomannan
Acidic galactan Aloin A and B (barbaloin) Lauric acid Vitamin B1 Aspartic acid Sodium
Mannans Isobarbaloin Myristic acid Vitamin B2 Glutamic acid Copper

Glucomannans 7-Hydroxyaloin Pentadecanoic acid Vitamin B6 Serine Zinc

Arabinogalactan Homonataloin Palmitic acid Vitamin C Histidine Chromium
Arabinans Chrysophanol Margaric acid Choline Lysine Selenium
Glucogalacto- Anthranol Stearic acid Vitamin D Threonine Aluminum
mannan
Cellulose Chrysophanol glucoside Palmitoleic acid Vitamin E Valine Magnesium
(a-tocopherol)
Pectins Aloesaponarin I & II Hexadecadienoic acid Folic acid Methionine Calcium
Polyuronide Helminthosporin Oleic acid Vitamin K Leucine Manganese
Sugars Tetrahydro-anthracene Linoleic acid Niacinamide Isoleucine Chlorine
glucoside
Glucose Anthracene Linolenic acid Enzymes Phenylalanine Sulfur
Mannose Emodin Organic Acids Amylase Tryptophane Iron
Arabinose Chromones* Malic acid Oxidase Histidine Lignins
Rhamnose Aloesin Succinic acid Catalase Hydroxyproline Lipids
Fructose p-Coumaroylaloesin Lactic acid Lipase Glutamine Cholesterol
Sucrose Isorabaichromone p-Coumaric acid Alkaline Proline Campesterol
phosphatase
Xylose Feruloylaloesin Salicylic acid Cellulase Alanine Sitosterol
Glucuronic acid Aloeresin A, B, C, & D Uronic acid Aliinase Tyrosin Triglycerides
Fucose Aloesone Uric acid Glyoxalase Cystine Lupeol
Galacturonic acid Flavonoids Cinnamic acid Lectins Asparigine Campherenol
Tannins Quercetin Fumaric acid Aloctin I & II Glycine Saponins
* Anthrones, anthraquinones, and chromones comprise most of the contents of the exudate which is obtained from the vascular-bundle sheath’s aloitic cells.
These compunds are also present in whole unfiltered leaf juice products.

and glutamic acid) (Boudreau and Beland 2006; Reynolds Carbohydrates

2004; Waller et al. 1978).
The putative biological activities attributed to the
internal and topical use of aloe vera leaf and inner leaf
juices include promotion of wound healing, anticancer,
antitumor, antidiabetic, antifungal, anti-inflammatory,
antioxidant, hypoglycemic, gastroprotective, liver-protective,
immunomodulatory, and kidney-protective effects (Hamman
2008). Many of the health benefits associated with aloe
vera leaf juice preparations have been attributed to the
polysaccharides contained in the inner leaf.
Following is a review of the primary compounds in
aloe vera leaf. For a more detailed review of aloe vera leaf
chemistry, see Park and Kwon (2006) and Reynolds (2004),
among others.
A significant amount of the solid components of aloe
vera leaf juice is made up of carbohydrates, including
polysaccharides, simple sugars, and a small amount of
oligosaccharides. Waller et al. (2004) estimated carbohydrates
to comprise 25% of the solid fraction of aloe vera leaf juice.
After complete hydrolysis, which converts all carbohydrates
into simple sugars, mannose and glucose accounted for up
to 85% of total sugars in the inner leaf juice, with a glucose/
mannose ratio of 1:1.3 (Waller et al. 1978). While glucose
occurs mostly in the free form, mannose exists largely in a
polymeric form.
Monosaccharides
D-Glucose is universally reported as the primary soluble
sugar in aloe vera leaf, comprising about 95% of the total

American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 19

monosaccharides (Femenia et al. 1999; Paez et al. 2000; juice products contain polysaccharides with molecular
Waller et al. 2004). Other sugars reported include fructose, weight distribution from few thousands to millions Da.
galactose (Paez et al. 2000); arabinose, and rhamnose (Bozzi Qiu et al. (2000) determined average molecular weight of
et al. 2007). In different aloe vera leaf juice products, glucose enzymatically modified aloe vera acetylated polysaccharide
content can range 4.0%–20.2% dry weight (Jiao et al. 2010). as 80 kDa.
The reported levels of monosaccharides in aloe vera inner The reported concentrations of polysaccharides in aloe
leaf juice vary between approximately 11% dry weight (Ni vera leaf juices vary widely. These variations can be due to
et al. 2004) and 27.81% dry weight (Femenia et al. 1999). the different processes used in juice manufacturing, seasonal
changes (Mandal and Das 1980a; Rodríguez-González et
Polysaccharides
al. 2011), differences of geographical location (Ni et al.
Polysaccharides in aloe vera leaf are characteristic components
2004), and variations in extraction and processing methods
of the plant and thus can be utilized for identification of
(Waller et al. 2004). Jiao et al. (2010) reported acetylated
the authenticity and quality of the products derived from
polysaccharide values from as low as 1.2% to 10.2%. Femenia
aloe vera leaf (e.g., aloe vera leaf juice and aloe vera inner
et al. (1999) reported approximately 34% polysaccharides in
leaf juice). Since the first isolation of polysaccharides
the depulped and extruded aloe vera “gel” (inner leaf juice).
from aloe vera, two distinct structural characteristics of the
According to Waller et al. (2004), the mean polysaccharide
polysaccharides were revealed. First, the polysaccharides
concentration in various batches of depulped inner leaf fillet
are mostly composed of polymerized mannose, with a
was 9.2 ± 2.5%. The highest value reported by the same
small percentage of glucose, connected mainly by β-(1→4)-
authors appears to be 24.1% of the solids in a depulped, non-
glycosidic linkages (Gowda et al. 1979). Second, mannose
decolorized whole leaf material that was not treated with
residues in the polysaccharides are acetylated at the second
cellulase. Decolorization reduced this amount to 23.1%.
(O-2), third (O-3), or sixth (O-6) oxygen atom positions,
The usage of exogenous enzymes in juice manufacturing,
with an average degree of acetylation being reported as 0.78
if not controlled, may lead to significant reduction of total
per mannose residue (Manna and McAnalley 1993). The
polysaccharide content. Pasteurization, decolorization, and
polysaccharides are considered the primary constituents of
prolonged storage of whole leaves typically also results in
medicinal interest in aloe vera leaf and inner leaf juices. In
reduced amounts of polysaccharides.
the aloe industry, the polysaccharides have been commonly
A variety of other polysaccharides have also been
referred to as “acemannan.”
characterized in minor amounts in the inner leaf of aloe vera,
Aloe vera acetylated polysaccharides have a wide range
including different subtypes of mannans, glucomannans,
of molecular weight distribution, exhibiting variations
and galactoglucomannans (Ni et al. 2004), arabinan and
in mannose content, degree of acetylation, and alcohol
arabinogalactan (Ni et al. 2004), galactan (Mandal and Das
solubility (Gowda et al. 1979). ‘t Hart et al. (1989) identified
1980b), small amounts of acidic poly- or oligosaccharides
the polysaccharides to be composed of mannose (83.7%-
containing glucuronic acid residues (Chang et al. 2011;
92.1%), glucose (3.2%-3.9%), galactose (3.8%-3.9%), and
Mandal et al. 1983), malic acid-acetylated carbohydrates
arabinose (0.9%-3.6%). The polysaccharides present in
veracylglucans A, B, and C (Esua and Rauwald 2006), and
the aloe vera inner leaf juice (“gel”) analyzed by Femenia
high-molecular-weight polysaccharide aloeride (Pugh et al.
et al. (1999) consisted of 81.6% mannose, 12.9% glucose,
2001). Other high-molecular-weight bioactive components
and small amounts of several other sugars (rhamnose,
of aloe vera leaf include maloyl glucans (e.g., veracylglucans)
fucose, arabinose, xylose, and galactose). Qiu et al. (2000)
and glycoprotein verectin (Davis et al. 1994; Esua and
described polysaccharides containing mannose, galactose,
Rauwald 2006; Yagi et al. 1998; Yagi and Takeo 2003). Some
and glucose in the ratio of 40:1.4:1.0. Aloe vera acetylated
researchers have pointed to pectic substance and galactans as
polysaccharides described by Chow et al. (2005) contained
the main components of the polysaccharide fraction in aloe
mannose (man), glucose (glc), galactose (gal), galacturonic
vera leaf (Mandal and Das 1980b; McConaughy et al. 2008;
acid (galA), fucose (fuc), arabinose (ara), and xylose (xyl)
Rodríguez-González et al. 2011). Pectic substance is the
with the man:glc:gal:galA:fuc:ara:xyl ratio of 120:9:6:3:2:2:1,
collective term for a group of closely related polysaccharides
with trace amounts of glucuronic acid and rhamnose.
often found associated with pectin, which include pectin,
The backbone consisted mainly of [→4)-β-Manp-(1→4)-β-
pectic acid, and arabinogalactan. The observation of pectic
Glcp-(1→] residues in an approximate man:glc ratio of 15:1,
substance as the main polysaccharide complex in aloe
with side-chain substitutions, on average, every 16 mannose
vera leaf appears to be limited to those products that
residues (Chow et al. 2005). Numerous other ratios of
have undergone significant bacterial deterioration prior
repeating glucose and mannose units have been reported
to or during processing and thus contain no acetylated
(Hamman 2008).
polysaccharides (Waller et al. 2004).
The majority of polysaccharides isolated by alcohol
precipitation from fresh aloe vera inner leaf have molecular Proteins, Glycoproteins, and Amino Acids
weights higher than 0.5 million Da. However, as noted,
partial breakdown of polysaccharides normally occurs during The protein content in aloe vera leaf ranges from 6.33% dry
juice manufacturing due to the activity of either exogenous weight in the skin to 8.92% dry weight in the gel (Femenia
or endogenous enzymes (Waller et al. 2004). Commercially et al. 1999). Some native enzymes were isolated from aloe
available aloe vera leaf juice and aloe vera inner leaf vera, including glyoxalase I and II (Norton et al. 1990),

20 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012


12a.
Figure 12 Constituents of Aloe vera leaf
12a. A proposed structure of Aloe vera acetylated polysaccharide
12b. Aloin, the primary compound of Aloe vera latex
glutathione peroxidase (Sabeh et al. 1993), and superoxide
dismutase (both in the rind and “inner gel”) (Sabeh et al.
1996). Yagi et al. (1997) reported on the glycoproteins in the
“gel” (inner leaf). Aken and Can (1999) gave an account
of the presence of lectins aloctin I and II in aloe vera leaf
and leaf juice (exudate included). Park and Son (2006)
reported on lectins, glycoproteins, and proteinase inhibitors
in aloe vera leaves. Das et al. (2011) reported the presence of
several novel proteins. At least 17 common amino acids were
detected in the inner leaf of aloe vera, of which arginine was
the most abundant, followed by asparagine, glutamic acid,
aspartic acid, and serine (Waller et al. 1978).
Lipids
The lipid content of aloe vera leaves ranges 2.71%–5.13%
dry weight (Femenia et al. 1999). Several steroids were
reported, including β-sitosterol, campesterol, cholesterol,
stigmasterol, lophenol, 24-methyl-lophenol, 24-ethyl-
lophenol, cycloartanol, 24-methylene-cycloartanol, and
lupeol (Tanaka et al. 2006; Waller et al. 1978). Yamaguchi
et al. (1993) detected a variety of alkanes and fatty acids in
freeze-dried “gel.”
Phenolic Compounds
Anthrones, anthraquinones, and chromones are the major
types of phenolic compounds in aloe vera leaf exudate (Park
and Kwon 2006) and are removed from IASC-certified juices
by decolorization. Anthrone C-glycosides aloins A and B
(also known as barbaloin) account for 10%–25% dry weight
of the exudate (Boudreau and Beland 2006). Aloins A and
B are diastereomers that can be easily separated by HPLC.
Aloin A has a 10S configuration at C-10 with a β orientation
(Figure 12b), while aloin B is the 10R diastereomer with an
α orientation. Chromones include aloesin (formerly called
aloeresin B) and aloeresin A, which, together with barbaloin,
are regarded as the most prominent constituents of aloe latex
(Park and Kwon 2006; Reynolds 2004). A number of other
anthrones, which are mostly derivatives of barbaloin, are
reported in aloe vera leaf exudate (e.g., see Okamura et al.
OH O OH
10 OH
HO
O
OH
OH
12b. OH
1997; Reynolds 2004). Among the anthraquinones, the most
notable is aloe-emodin. For a more detailed review of other
compounds occurring in aloe vera leaf exudate, see Park
and Kwon (2006) and Reynolds (2004). An isocoumarin,
feralolide, was found in the leaves of aloe vera (Choi et al.
1996).
Organic Acids
As much as 75% of the solids in aloe vera leaf and inner leaf
juices may consist of organic acids, metal ions, and chloride
(Waller et al. 2004). Malic acid is the most abundant organic
acid in aloe vera inner leaf juice, with a content of 11.1%–
40.4% weight of the solid fraction (Jiao et al. 2010). Since
malic acid is prone to bacterial degradation to lactic acid by
Lactobacillus spp., content of malic acid can be used as a
marker for “freshness” and quality of aloe vera leaf and inner
leaf juices. IASC aloe vera leaf juice products commonly
contain lower malic acid levels than aloe vera inner leaf
juice products (Waller et al. 2004).
Citrate, isocitrate, and isocitrate lactone have been
found in higher amounts in the outer portion of aloe vera leaf
(Waller et al. 2004). Hence, the presence of high amounts
of isocitrate is used as a negative marker for aloe vera inner
leaf juice products. Citric acid can also be added to products
as an acidulant or preservative. Other organic acids that
may be present in the juices include quinic acid (Paez et
al. 2000), fumaric, succinic, and acetic acids. Fumaric acid
and succinic acid are products of the citric acid cycle in the
plant. If this cycle is allowed to continue during storage,
enzymatic degradation of the polysaccharides can occur,
resulting in the increase of organic acid concentrations.
Another important outcome of degradation of aloe vera
polysaccharides is their deacetylization, which results in
the production of acetic acid (Bozzi et al. 2007). Thus,
estimation of the levels of these compounds can be used to
assess processing and storage conditions.
Minerals
According to Rajasekaran et al. (2005), the following
elements occur in the “gel layer” (inner leaf) of aloe vera
leaf, in the order of decreasing concentrations: Fe, Mn, K,
Zn, V, Na, Mg, Cu, Cr, Ca, and Pb. Conversely, Ca2+ was
the most abundant metal ion in aloe vera “gel” (inner leaf
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 21
juice) analyzed by Femenia et al. (1999). Aloe vera leaf juice
products commonly have a relatively high mineral content,
as compared with aloe vera inner leaf juice products (Waller
et al. 2004).
A na ly t i ca l
The following methods are provided for the identification
and quality assessment of aloe vera leaf and aloe vera
leaf juices (liquid and dry). The high performance thin
layer chromatography (HPTLC) is predominantly used
to confirm the identity of aloe vera leaves used for the
manufacturing of juice products, including the detection of
the prominent alternate species in trade. The method can
also be used to establish the presence of exudate compounds
(such as aloins A and B). However, as shown in the
chromatograms provided, the HPTLC methodology is not
applicable for the identification of processed juice products.
The high performance liquid chromatography (HPLC)
method is primarily used for the quantitation of aloins A and
B in products and can be used to ensure conformity with
the IASC limits for these compounds. For screening of the
presence of maltodextrin, (e.g., to confirm its absence if not
stated in labeling), a simple and inexpensive Maltodextrin
Assay is provided. For detailed detection and quantitation of
the primary compounds of interest in aloe leaf and its juices
(i.e., acetylated polysaccharides), including for compliance
with IASC criteria, see “Proton Nuclear Magnetic Resonance
Spectrometry (1H NMR)” in the Analytical section of the
Aloe Vera Leaf Juice monograph.
High Performance Thin Layer Chromatography
(HPTLC) for the Identification of Aloe Vera
Leaf
This HPTLC method was based on the methods for Barbados
and Cape Aloes from the European Pharmacopoeia 6.0 with
variations to sample preparations to allow for the analysis of
various leaf products. The chromatographic conditions are
also consistent with those provided in the pharmacopoeias of
China and India. This method can be used for the detection
of aloins A and B and aloe-emodin in raw materials and
finished products, for identifying Aloe vera leaves, and for
distinguishing crude aloe vera preparations and finished
aloe vera leaf products from A. arborescens and A. ferox. The
characteristic fingerprint of Aloe vera is best visualized at UV
366 nm, however, detection of the band that differentiates
Aloe vera from the Cape Aloes (A. ferox) is best visualized in
white light. Aloe arborescens is best visualized in UV 366 and
is clearly distinguished from A. vera and A. ferox. The various
aloe juice preparations show considerable differences from
the crude leaf due to the filtration and processing to remove
compounds mainly located in the exudate.
Sample Preparation
Fresh whole leaf: Cut into 2 to 3 mm squares, dry in a 30
°C oven for 36 hours, and powder in an analytical mill. Mix
0.50 g of the powder with 10 mL of methanol, sonicate for
22 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012
15 min, and centrifuge or filter. Use the supernatant (or
filtrate) as the test solution.
Fresh inner leaf: Fillet the inner leaf (cut away and discard
the rind) and cut the inner gelatinous mass into 2-3 mm
squares. Dry the material in a 30 °C oven for 36 hours and
powder in an analytical mill. Mix 0.50 g of powder with
10 mL of methanol, sonicate for 15 min, and centrifuge or
filter. The supernatant or filtrate is used as the test solution.
Processed inner leaf and juices: Dry about 25 mL of the
material in a 30 °C oven for 36 hours. Mix 0.50 g of the
material with 10 mL of methanol, sonicate for 15 min, and
centrifuge or filter. Use the supernatant (or filtrate) as the
test solution.
Extracts, powders, and tablets: Mix 0.50 g with 10 mL of
methanol, sonicate for 15 minutes, and centrifuge or filter.
Use the supernatant (or filtrate) as the test solution.
Standards Preparation (optional)
Aloin A: Dissolve 1 mg of aloin A in 10 mL of methanol.
Aloin B: Dissolve 1 mg of aloin B in 10 mL of methanol.
Aloe-emodin: Dissolve 1 mg of aloe-emodin in 10 mL of
methanol.
Reagent Preparation
Potassium hydroxide (KOH) reagent: Dissolve 20 g of KOH
pellets in 200 mL methanol in an ice bath.
Chromatographic Conditions
Stationary Phase:
HPTLC plates 10 × 10 cm or 20 × 10 cm silica gel 60
F254.
Mobile Phase:
Ethyl acetate:methanol:water (100:17:13).
Sample Application:
Apply 5 mL of test solution(s) and 2 mL of each reference
standard as an 8 mm band with a minimum of 2 mm
distance between bands. Application position should be 8
mm from lower edge of plate.
Development:
10 × 10 cm or 20 × 10 cm Twin Trough Chamber
(CAMAG or equivalent), lined with filter paper,
saturated for 20 minutes with 5 or 10 mL, respectively,
of developing solvent in each trough. Developing
distance is 70 mm from the lower edge of the plate. Dry
the plate in a stream of cold air for 5 minutes.
Detection:
a) Examine the plate under UV 366 nm.
b) Dip the plate in KOH reagent and then heat at 110
°C for 5 min. Examine under UV 366 nm.
c) Dip the plate in KOH reagent. Examine under
white light.
Results:
Compare with the chromatograms provided (Figures
13 and 14).
Figure 13a HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (UV 366 nm)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively) 0.43) an orange-brown fluorescent zone due to aloin, and a faint,
show zones of orange-brown fluorescence (Rf 0.43) and aloe diffuse zone of yellow fluorescence (Rf 0.79) due to aloe emodin. A
emodin (lane 2) shows a zone of yellow fluorescence (Rf 0.79). The zone of red fluorescence due to chlorophyll is seen at the solvent
chromatograms obtained with the test solutions show in the lower front (not detectable in the inner leaf samples on lanes 12 and 13).
part (Rf 0.30) a light blue fluorescent zone, in the central part (Rf

Figure 13b HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (UV 366 nm, derivatized)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively) faint, diffuse zone of red fluorescence (Rf 0.79) due to aloe emodin
show zones of yellow fluorescence (Rf 0.43) and aloe emodin can be detected (this is detected more clearly in all of the samples
(lane 2) shows a zone of dark red fluorescence (Rf 0.79). The in the pre-derivatized UV 366 nm image above, 13a). A zone of red
chromatograms obtained with the test solutions show in the lower fluorescence due to chlorophyll is seen at the solvent front (not
part (Rf 0.30) a light blue fluorescent zone and in the central part detectable in the inner leaf samples on lanes 12, 13 and 14).
(Rf 0.43) a yellow fluorescent zone due to aloin. In some samples a

American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 23

Figure 13c HPTLC chromatogram of Aloe vera leaf and leaves of other Aloe species (white light, derivatized)
Discussion of the chromatogram
The standards aloin A and aloin B (lanes 1 and 2, respectively) emodin. The Aloe vera and unknown Aloe test samples show in
show brown zones (Rf 0.43) and aloe emodin (lane 2) shows a visible light a violet-brown zone (Rf 0.39) just below the zone due
red-brown zone (Rf 0.79). The chromatograms obtained with the to aloin. The Aloe ferox and Aloe arborescens samples (lanes 8, 9,
test solutions show in the central part (Rf 0.43) a brown zone due 14 and 15) are lacking this zone. The presence of this zone can be
to aloin and a faint, diffuse red-brown zone (Rf 0.79) due to aloe used to differentiate Aloe vera from the Cape Aloes (Aloe ferox).

Lane 1: Aloin A
Lane 2: Aloe emodin, aloin B
Lane 3: Aloe vera fresh whole leaf
Lane 4: Aloe vera fresh outer leaf
Lane 5: Aloe vera fresh whole leaf
Lane 6: Aloe vera fresh whole leaf
Lane 7: Aloe vera fresh whole leaf
Lane 8: Aloe ferox fresh whole leaf
Lane 9: Aloe arborescens fresh whole leaf
Lane 10: Aloe vera fresh inner leaf
Lane 11: Aloe vera fresh inner leaf
Lane 12: Aloe vera fresh inner leaf
Lane 13: Aloe vera fresh inner leaf
Lane 14: Aloe ferox fresh inner leaf
Lane 15: Aloe arborescens fresh inner leaf

24 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

Figure 14a HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (UV 366 nm)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and
23) show zones of orange-brown fluorescence (Rf 0.43) and aloe
emodin (lanes 3 and 24) shows a zone of yellow fluorescence (RF
0.79). The chromatograms obtained with most of the test solutions
show in the lower part (Rf 0.30) a light blue fluorescent zone.
This zone is not detected in the Aloe vera juice, tablets, or flakes
(lanes 11, 20, and 21, respectively). In the central part (Rf 0.43) an
orange-brown fluorescent zone due to aloin is detected in most
samples, except for some of the processed Aloe vera products
Figure 14b HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (UV 366 nm, derivatized)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and
23) show zones of yellow fluorescence (Rf 0.43) and aloe emodin
(lanes 3 and 24) shows a zone of dark red fluorescence (Rf 0.79).
The chromatograms obtained with most of the test solutions show
in the lower part (Rf 0.30) a light blue fluorescent zone. This zone
is not detected in the Aloe vera juice and flakes (lanes 11 and
21, respectively). In the central part (Rf 0.43) a yellow fluorescent
zone due to aloin is detected in most samples, except for some
of the processed Aloe vera products (juices, tablets, and flakes
on lanes 11, 12, 20, and 21, respectively). A faint, diffuse zone of
(juices, “gel,” tablets, and flakes on lanes 11, 12, 14, 20, and 21,
respectively). A faint, diffuse zone of yellow fluorescence (Rf 0.79)
due to aloe emodin is seen in most samples, except for some of
the processed Aloe vera products (juices, “gel,” tablets, flakes,
and powder on lanes 11, 12, 14, 19, 20, and 21, respectively), and
the Aloe ferox juice (lane 16). A zone of red fluorescence due to
chlorophyll is seen at the solvent front in the unprocessed leaf
samples, the Aloe ferox juice (lane 15), and the two Aloe vera leaf
extracts (lanes 17 and 18).
dark red fluorescence (Rf 0.79) due to aloe emodin is detected in
few samples. This zone of aloe emodin is more easily detected in
the pre-derivatized UV 366 nm image (Figure 14a). A zone of red
fluorescence due to chlorophyll is seen at the solvent front in the
unprocessed leaf samples, the Aloe ferox juice (lane 15), and the
two Aloe vera leaf extracts (lanes 17 and 18).
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 25
Figure 14c HPTLC chromatogram of Aloe vera leaf, leaves of other Aloe species, and Aloe spp. products (white light, derivatized)
Discussion of the chromatogram
The standards aloin A (lanes 1 and 22) and aloin B (lanes 2 and 23)
show brown zones (Rf 0.43) and aloe emodin (lanes 3 and 24) show
a red-brown zone (Rf 0.79). The chromatograms obtained with the
test solutions show in the central part (Rf 0.43) a brown zone due
to aloin This zone is non-detectable in some of the processed Aloe
vera products (juices on lanes 11 and 12, tablets on lane 20, and
flakes on lane 21) or in the Aloe ferox juice (lane 16). Many of the
samples also show a faint, diffuse red-brown zone (Rf 0.79) due
to aloe emodin. Again, this zone is not detectable in some of the
processed Aloe vera products (juices on lanes 11 and 12, “gel”
on lane 14, powder on lane 19, tablets on lane 20, and flakes on
lanes 21) or in the Aloe ferox juice (lane 16). The Aloe vera and
unknown Aloe fresh whole leaf test samples, Aloe vera gel (lane
13), and Aloe vera whole leaf powder (lane 19) show in visible light
a violet-brown zone (Rf 0.39) just below the zone due to aloin. This
zone is non-detectable in the other processed Aloe vera products.
The Aloe ferox and Aloe arborescens samples (lanes 8, 9, 14 and
15) are lacking this zone. The presence of this zone can be used to
differentiate Aloe vera from the Cape aloes (Aloe ferox).

Lane 1: Aloin A Lane 16: Aloe ferox drink concentrate

Lane 2: Aloin B Lane 17: IASC-certified Aloe vera inner leaf juice concentrate
Lane 3: Aloe emodin (200x; capsules)
Lane 4: Aloe vera fresh whole leaf Lane 18: IASC-certified Aloe vera leaf juice concentrate
Lane 5: Aloe vera fresh outer leaf Lane 19: IASC-certified Aloe vera whole leaf powder (100x)
Lane 6: Aloe vera fresh whole leaf Lane 20: IASC-certified Aloe vera tablets (whole leaf)
Lane 7: Aloe vera fresh whole leaf Lane 21: IASC-certified Aloe vera inner leaf flakes (200x)
Lane 8: Aloe vera fresh whole leaf Lane 22: Aloin A
Lane 9: Aloe ferox fresh whole leaf Lane 23: Aloin B
Lane 10: Aloe arborescens fresh whole leaf Lane 24: Aloe emodin
Lane 11: Aloe vera leaf juice
Lane 12: Aloe vera inner leaf juice
Lane 13: Aloe vera inner leaf juice
Lane 14: Aloe vera inner leaf juice
Lane 15: Aloe ferox juice

26 American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012

International Status
United States
Cosmetic Product: Aloe vera leaf and inner leaf juice is
included in many aloe vera skin care products.
Food Additive: Aloe vera (cited as aloe) is approved as a
flavoring agent or in conjunction with flavors only.
Dietary Supplement Ingredient: Aloe vera products designed
for oral consumption can be labeled and marketed as a
dietary supplement product (USC 1994), requiring FDA
notification and substantiation to support structure/function
claim statements.
Australia
“Gel”: May be used as an active ingredient in ‘Listed’
medicines in the Australian Register of Therapeutic Goods
(ARTG) for supply in Australia. Specific Indications:
Aids the relief of sunburn, insect bites, chafing rashes and
irritation of other sensitive skin areas. For the first-aid care
of minor burns: cool the affected area with cold water or
ice first for a suitable period before applying gel. Medical
advice should be sought for the care of more serious burns
(TGA 2009a).
Juice and Juice Concentrate: Some aloe vera juice and juice
concentrate beverages are viewed as “non-traditional foods”
and not as “novel foods” (FSANZ 2009). There are some
listed medicines described as “Aloe vera drinking gel” or
as “Aloe vera juice.” Standard Indications: Aids digestion;
helps maintain healthy digestive function; aids, assists
or helps in the maintenance or improvement of general
wellbeing (TGA 2009b). Specific Indications: Acts as a
general tonic, helps to maintain healthy digestive function,
and has a cleansing effect on the bowel (TGA 2009c).
Canada
“Leaf Gel” (Inner Leaf Juice): Included as a Natural
Health Product (NHP) active ingredient, requiring pre-
marketing authorization and product license for over-
the-counter (OTC) human use. Quality: Must comply
with the minimum specifications outlined in the current
NHPD Compendium of Monographs (NHPD 2007b).
Pharmacopoeial standards currently accepted by the
NHPD are the British Pharmacopoeia (BP), European
Pharmacopoeia (PhEur) and United States Pharmacopeia
(USP). If no monograph exists in the currently accepted
pharmacopoeia, other internationally recognized standards
can be used such as World Health Organization (WHO)
(NHPD 2007a). Compendial Indications (Topical):
Preparations containing at least 10%–70% leaf gel are (1)
traditionally used in herbal medicine to treat minor burns,
including sunburns; traditionally used to assist in wound
healing; and (2) traditionally used in herbal medicine to
assist healing of minor wounds such as cuts and burns, and
minor skin irritations (NHPD 2008).
European Community
“Gel” (Inner Leaf or Inner Leaf Juice): As of June 2012,
aloe vera “gel” was listed as a low priority in the inventory
of herbal substances for assessment by the European
Medicines Agency (EMA 2012).
The European Commission database on cosmetic ingredients
(CosIng 2012) reports that aloe vera leaf materials are
approved for a variety of functions, predominantly for skin
conditioning.
India
Leaf Pulp: The leaf pulp of Aloe barbadensis (syn. A. vera)
is used in the Unani Systems of Medicine. Indications:
To treat bawaseer (piles), sual-nazla (cough and cold), and
wajiul mafasil (rheumatism) (CCRUM 1992).
Japan
Leaf Gel, Juice, and Various Extractives: Aloe vera juice is
regulated as a food beverage product. It may not contain
more than 0.60 mg/kg of benzoic acid. Various forms of
aloe vera and extracts thereof are used as components of
functional food products or in Foods for Specified Health
Use (FOSHU) such as in fortified waters and fermented
yogurt drinks (Yamaguchi 2004).
South Korea
Edible Aloe Concentrate and Edible Aloe Gel: Regulated
as food products (KFDA 2004). Juice or concentrate from
the gel or dried and powdered gel containing not less than
30 mg/g of aloe total polysaccharides are permitted the
following health claims at the specified daily intake (dosages
expressed as the amount of aloe polysaccharides): for skin
health (100–420 mg), for colon health (110–125 mg), for
immune enhancement (100–290 mg).
Whole Leaf, Concentrated or Pulverized: After removal of
the inedible parts, the pulverized whole leaf or pulverized
concentrate from the whole leaf, containing 2.0-50 mg/g
anthraquinones (as anhydrous barbaloin), is permitted
the following health claim at the specified daily intake
(dosage expressed as the amount of aloe polysaccharides):
“smoothing the evacuation” (20-30 mg).
World Health Organization
“Gel” (Inner Leaf Juice): A monograph for the topical
use of the colorless mucilaginous gel obtained from the
parenchymatous cells in the fresh leaves of Aloe vera is
published in the WHO Monographs on Selected Medicinal
Plants. Medicinal Uses Supported by Clinical Data:
None. Several health benefits are listed including wound
healing, anti-inflammatory, and as a burn treatment (WHO
1999). Posology: Fresh gel or preparations containing 10%-
70% fresh gel (WHO 1999).
Note: This review only addresses the international status of the aloe vera leaf,
aloe vera inner leaf, and aloe vera leaf juice products, not the concentrated
exudate (latex), included in most international pharmacopoeias, or other
aloe species.
American Herbal Pharmacopoeia® • Aloe Vera Leaf • 2012 27
Aloe Vera Leaf Juice
Aloe vera (L.) Burm. f.
Introduction
Consistent with the terminology of the International Aloe
Science Council (IASC), aloe vera leaf juice is produced
from the entire leaf, typically involving enzymatic treatment,
and processed in a manner that meets IASC criteria for
quality, including limits for phenolic compounds (aloins A
and B) and acetylated polysaccharide content, among other
specific criteria (Table 1). This definition of “aloe vera leaf
juice” is used to distinguish this type of products from those
prepared from the inner leaf only, predominantly for the
purpose of labeling.
Nomenclature
Pharmacopoeial Definition
Aloe vera leaf juice consists of the liquid derived from the
entire leaves of Aloe vera (L.) Burm. f., or the dry powdered
concentrate of the liquid. The juice contains not less than
5% dry weight of acetylated polysaccharides, determined by
proton nuclear magnetic resonance spectrometry.
Identification
Macroscopic Identification
Aloe vera leaf juice is a cloudy to transparent liquid.
Organoleptic Characterization
Charcoal-Filtered Liquid (single strength)
Color: Colorless to caramel-colored.
Aroma: Odorless to mildly vegetative.
Taste: Tasteless to slightly bitter.
Charcoal-Filtered Dry Concentrate (flakes or powder)
Color: Beige.
Aroma: Odorless to mildly vegetative.
Taste: Tasteless to slightly bitter.
Note: The organoleptic characteristics of aloe vera leaf juice products can
vary considerably, depending on the processing techniques and additives
used.
28 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012
Commercial Sources
and Handling
In the production of aloe vera leaf juice, special care must be
taken to preserve the content of acetylated polysaccharides,
which can readily degrade due to prolonged storage,
bacterial fermentation, and elevated temperatures. The
quality of commercial aloe vera leaf juice is strongly
dependent on processing and storage conditions. Enzymatic
and thermal degradation and bacterial fermentation may
affect the quality and decrease the value of the final product.
For a more thorough discussion of processing involved in
the production of aloe vera leaf juice, see the Aloe Vera Leaf
monograph.
Due to safety concerns in relation to aloins A and B (CIR
2007), the aloe vera industry began using activated charcoal
in the manufacturing of aloe vera leaf juice in the process
called “decolorization.” This practice can consistently
reduce the concentration of phenolic compounds in aloe
vera leaf juice products. The total content of aloins A and B
must not be more 10 ppm in IASC-compliant single-strength
aloe vera leaf juice raw materials and finished products
intended for oral consumption. For other parameters of the
IASC certification process, see Table 1.
Table 1 IASC certification requirements for aloe vera leaf juice
Compound Certification Requirement
Acetylated
≥ 5% dry weight
polysaccharides
Glucose Present
≤ 10 ppm in 1.0% aloe vera leaf juice
solids solution, analyzed by HPLC or
Aloin A & B
other fit-for-purpose methodology
approved by the IASC
Must be listed on label and analysis
Maltodextrin
must meet label claims.
Solids ≥ 1.0% in single-strength leaf juice
Ash ≤ 40%
Constituents
The solids content of aloe vera leaf juice varies depending
on the processing techniques. The solids fraction contains
mainly carbohydrates, organic acids, and mineral salts
(Waller et al. 2004), with most of the other components
removed during processing. The typical composition of
aloe vera leaf juice is provided in Table 2. For a more
detailed discussion of aloe vera polysaccharides and other
constituents, see the Aloe Vera Leaf monograph.
Table 2 Typical constituent profile of aloe vera leaf juice as
determined by 1H-NMR

Component Content (% dry weight)

Acetylated mannan 5.36

Glucose 6.76

Malic acid 15.28

Lactic acid 0.09

Citric acid 9.29

Pyruvic acid 0.18

Sorbate 0.17

Benzoate 0.29 1a.

Isocitrate 6.06

Isocitrate lactone 3.40

Acetic acid 0.21

Succinic acid 0.00

Formic acid 0.00

Fumaric acid 0.00

Ethanol 0.00

Maltodextrin 0.00

A na ly t i ca l 1b.

Maltodextrin Assay Figure 1 Maltodextrin test

Maltodextrin is an acceptable carrier used in spray-dried of
aloe vera leaf juice products (typical ratio 1:1). Maltodextrin
is also reported as one of the most prevalent adulterants of
aloe vera leaf juice products used to artificially enhance
polysaccharide values. This colorimetric assay can be
used as an initial screening tool for identifying potentially
adulterated aloe vera juice ingredients. The following assay
was modified from a commonly known starch detection
method. The assay should be performed with a control that
is free of maltodextrin.
1a. Aloe vera leaf juice with and without maltodextrin (left to right:
0%, 10%, 25%, 50% weight of maltodextrin) prior to color
reaction with iodine.
1b. Aloe vera leaf juice with and without maltodextrin showing
color reaction after the addition of iodine.
Reagents
Iodine Solution: In 250-mL volume flask, dissolve 10 g
potassium iodide (KI) in 25 mL water. Add 3.175 g iodine
(I2) and dilute to 250 mL.

Sample Preparation Detection

Liquid Samples: Add 10 mL of the liquid sample being Add 1–3 drops of Iodine Solution to the samples being
tested to an empty test vial. tested. Cap the vials and invert one time, or swirl.
Dry Samples: Reconstitute in an appropriate amount of Results
water to achieve single-strength juice (refer to concentration In the presence of maltodextrin, the solution will turn
stated on the material labeling documentation). Add 10 mL medium- to dark-brown or purple to black, depending on
of the liquid to an empty test vial. the amount of maltodextrin present (Figure 1).
Standard Preparation (optional)
Add 10 mL of aloe vera leaf juice reference material
(e.g., AHP-Verified aloe vera leaf juice), reconstituted in
appropriate amount of water to achieve single-strength juice,
to an empty test vial.

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 29

High Performance Liquid Chromatography
(HPLC) for the Quantitation of Aloins A and B
in Aloe Vera Leaf Juice
The following HPLC procedure can be used to quantify
aloins A and B in aloe vera leaf juice products to meet
IASC requirements of not more than 10 ppm of total aloins
in single-strength raw materials and finished product. The
method was subjected to a single-laboratory validation
according to guidelines of AOAC International. The
following preparations were analyzed: processed and
unprocessed aloe vera leaf juice (1×), aloe vera leaf juice
powder (100×), aloe vera inner leaf juice powder (200×),
and capsules with powdered concentrate. The limit of
detection (LOD) and limit of quantitation (LOQ) were
determined according to the guidelines of the International
Union of Pure and Applied Chemistry (IUPAC).
Sample Preparation
Liquid Samples
Processed samples (e.g., decolorized aloe vera leaf juice):
Directly filter approximately 1 mL of the sample through 0.2
µm PTFE into a HPLC vial for analysis.
Unprocessed samples (e.g., non-decolorized aloe vera leaf
juice): Dilute (1:1 v/v) with 0.1% acetic acid in methanol.
Filter approximately 1 mL of the diluted solution through
0.2 µm PTFE into a HPLC vial for analysis.
Powdered Samples
Weigh 100.0 ± 1.0 mg of the powdered juice concentrate
into a 15-mL conical centrifuge tube. Add 1.0 mL 0.1%
acetic acid solution in water and sonicate for 5 min three
times; vortex 30 seconds between intervals. The supernatant
is then filtered through 0.2 µm PTFE filter into an HPLC
vial.
Capsules
Combine and homogenize the contents of 20 capsules and
follow instructions for powders above.
Note: For samples that are highly concentrated or viscous, add 1 mL of 0.1%
acetic acid solution in water followed by adding 1 mL of 0.1% acetic acid in
methanol. Sonicate for 5 min three times, vortexing for 30 seconds in the
sonication intervals. Centrifuge at 5000 rpm (4500× g) for 3 min. If samples
are still concentrated or viscous after the 1:1 dilution of 0.1% acetic acid in
water and methanol, add another 1 mL of 0.1% acetic acid solution in water,
and sonicate, vortex, and centrifuge the sample again prior to filtering and
aliquoting it to a HPLC vial.
Standards Preparation
Aloin A stock solution (0.1 mg/mL): Weigh 10.0 ± 0.1 mg of
aloin A into a 100-mL volumetric flask, add approximately
80 mL of methanol, mix thoroughly, and fill to mark with
methanol.
Aloin B stock solution (0.1 mg/mL): Weigh 10.0 ± 0.1 mg
of aloin B to a 100-mL volumetric flask, add approximately
80 mL of methanol, mix thoroughly, and fill to mark with
methanol.
Aloin A Calibration Curve
Calibration standards are prepared by serial dilution, as in
Table 3.
A mixed standard of 25 µg/mL aloins A and B can be
employed for quality control (QC) purposes. Prepare the
QC standard by diluting 1 mL of the 0.1 mg/mL aloin A
stock solution and 1 mL of the 0.1 mg/mL aloin B stock
solution in 2 mL of 0.2% acetic acid in water.
Instrumentation
The HPLC system must be equipped with a diode-array
detector or UV detector capable of monitoring at 357
nm, sample injection system, pump capable of delivering
constant flow up to 600 bar, temperature-controlled column
compartment, and computing data processor. Equilibrate
the HPLC system with the mobile phase prior to analysis.
Reagents
Acetic acid, glacial: ACS reagent grade or equivalent.
Methanol: HPLC grade or equivalent.
Acetonitrile: HPLC grade or equivalent.
Water: HPLC grade or equivalent.
Mobile phase A: 0.1% volume acetic acid in water. Prepare
by adding 1 mL of glacial acetic acid to 999 mL of 0.2
µm-filtered Nanopure water. Sonicate for 10 minutes.
Mobile phase B: 0.1% volume acetic acid in acetonitrile.
Prepare by adding 1 mL of glacial acetic acid to 999 mL of
acetonitrile. Sonicate for 10 minutes.

Table 3 Calibration standards for aloin A calibration curve

Volume of 0.1 mg/ Volume of 25-µg/ Volume of 5-µg/ Volume of 1-µg/ Volume of 0.2%
Standard label Volume of
ml stock standard mL standard mL standard mL standard acetic acid in
(µg/mL) methanol (mL)
solution (mL) (mL) (mL) (mL) water (mL)

Std-50 0.5 – – – 0.5 –

Std-25 1 – – – 2 1

Std-10 1 – – – 5 4

Std-5 – 1 – – 2 2

Std-1 – – 1 – 2 2

Std-0.2 – – – 1 2 2

30 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012

Stability and Storage of Preparations Table 4 Mobile phase gradient for high-performance liquid
Refrigeration of test samples at 4 °C will maintain a relative chromatography analysis of aloins A and B in aloe vera leaf juice
stability of aloins for up to 2 weeks.
Time, min Mobile phase A, % Mobile phase B, %
Chromatographic Conditions
0.0 83 17
Column:
Phenomenex Kinetex 2.6 µm C18 100 x 4.6 mm. 8.0 83 17
Column Temperature: 12.0 0 100
30 ºC.
13.0 0 100
Mobile Phase:
Gradient (see Table 4). 14.0 83 17
Flow Rate:
18.0 83 17
1.850 mL/min.
Injection Volume: Do not proceed with test sample analysis unless
15 mL. reproducibility and resolution requirements are met.
Detection: Inject a reagent blank followed by the calibration standards
357 nm. of aloin A (n = 6) as listed in Table 3.
Run Time: Inject up to 10 test samples followed by a blank and then
18 min. the QC standard. Repeat as necessary until all test samples
Elution Order: are run. It is recommended that samples be prepared in
Aloin A: ~6.9-7.1 min. triplicate. At the end of the run, re-inject the 25 ppm QC
Aloin B: ~5.4-5.6 min. standard. The QC standard injections should meet the
reproducibility criteria stated above.
Procedure and System Suitability
a) Equilibrate the HPLC system with the mobile phases As part of system suitability requirements, use the following
prior to analysis. formulas to verify that the capacity factor, tailing factor, and
signal-to-noise criteria of aloin A are met.
b) Perform a blank injection to condition the column.
Capacity Factor (k’):
c) Inject 50 ppm aloin A standard to ensure the standard is
The capacity factor for aloin A must be greater than 2.0.
correctly prepared and system is operational. The aloin A
The formula to use is as follows:
peak should elute at the time stated above and there should
be no other peaks present. Equation 2
d) Inject the QC standard (25 µg/mL mixed standard of k’ = (tA – t0) / t0
aloin A and aloin B) five times to ensure the reproducibility
and resolution requirements are met. where:
tA = the retention time of aloin A;
Reproducibility:
t0 = the retention time of the void volume.
The relative standard deviation (RSD) of each aloin
peak (aloin A and aloin B) areas and retention times Tailing Factor (T):
must be less than or equal to 2.0%, when using five The tailing factor should be less than 1, at 5% of peak
or fewer (n ≤ 5) replicate injections, or less than or height. The formula to use is as follows:
equal to 2.5%, when using six replicate injections. If Equation 3
reproducibility is not met, do not proceed with the
T = W0.05 / 2 * a
analysis.
Resolution (Rs): where:
The peaks of aloin A and aloin B should be resolved at W0.05 = peak width at 5% of the peak height;
greater than 1.5 when calculated based on peak width at a = distance from the leading edge of the peak to
tangent from any neighboring peaks using the following the midpoint.
formula:
Signal-to-Noise Ratio (S/N):
Equation 1 The signal-to-noise ratio should be monitored for the 0.2
Rs = 2(tB – tA) / (WA + WB) ppm standard. The noise is calculated by multiplying
the standard deviation of the linear regression of the
where: drift by 6 (closest range to the aloin A peak is manually
tA and tB = the retention times of aloin A and aloin B, selected in the software). The formula to use is as
respectively; follows:
WA and WB = the widths of the peak bases of aloin A and
Equation 4
aloin B, respectively.
S/N = height of the peak / noise of closest range

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 31

 mAU
40
Aloin B Aloin A
50 ppm QC standard
3a.
40
30

30
20 aloin A

aloin B
20
10

0 10

-10 0

0 2 4 6 8 10 min 0 2 4 6 8 10

Figure 2 HPLC chromatogram of aloins A and B standards 3b.

40

Calculations 30

Obtain the integrated area (I) values of aloin A in the

calibration standards and aloins A and B in the analyzed 20

samples. Construct a plot of concentration (µg/mL) of the

aloin A calibration standard (x-axis) against the individual
10
aloin A
aloin B

peak area (y-axis). Calculate the slope (m), intercept (b) and 0

correlation coefficient (R2) of the best-fit line. 0 2 4 6 8 10

The concentration of aloin A or aloin B in the prepared Figure 3 Typical HPLC chromatogram of aloe vera leaf juice
sample is calculated by the following equation:
3a. Non-decolorized aloe vera leaf juice.
Equation 5 3b. Aloe vera leaf juice after decolorization.
Cv = (Ialoin A/B – b) / m
aloe vera leaf juice of 10 mg/mL (1% w/vol). Lower strengths
where: indicate that other agents (e.g., carriers) have been added
Cv = concentration of aloin A or B in the sample vial, µg/ during the drying process. The following formula can be
mL; used to determine the total aloin concentration in single-
Ialoin A/B = integrated area of the aloin A or B peak in the strength preparations:
sample; Equation 8
b = intercept of the calibration curve;
m = slope of the correlation curve. C1x = Cs * Ds * S

Determine the concentration of total aloin (Ct, µg/mL) in

the sample vial by adding individual aloin concentrations: where:
C1x = concentration of total aloin, ppm or µg/mL, in single-
Equation 6
strength aloe vera leaf juice;
Ct = Cv of aloin A + Cv of aloin B
Cs = total aloin concentration in the sample, determined in
the Equation 7 above, µg/mg;
For powdered material, the concentration of aloin A and Ds = dilution factor, based on the “strength” of the product,
B in the original sample is calculated by the following estimated by the following equation:
equation:
Equation 9
Equation 7 Ds = 100 / x
Cs = Ct * V1 * D / Ms
where:
x = “strength” of the powdered concentrate stated in
where:
the documentation accompanying the material;
Cs = concentration of total aloin in the original sample, µg/
S = standard concentration of juice solids in single-strength
mg;
aloe vera leaf juice = 10 mg/mL.
Ct = concentration of total aloin A and B in the sample vial,
µg/mL, determined in the Equation 6 above; Aloe vera leaf juice can also be marketed as liquid
V1 = volume of the prepared sample, mL (here 1 mL); concentrates, e.g., 5×, etc. For liquid material, first obtain
D = dilution factor if applicable; the “strength” value of the liquid and determine the total
Ms = mass of original sample, mg. content of aloins A and B in single-strength juice by the
following equation:
To determine total aloin concentration in single-strength
juice, first obtain the “strength” value of the juice, typically Equation 10
stated on the label or in the documentation accompanying C1x = Ct * D * Ds
the material, such as certificate of analysis (COA). Pure aloe
where:
vera leaf juice powder is usually designated as having 100×
C1x = total concentration of aloins, ppm or µg/mL, in single-
strength, based on the standard value of the solids content in
32 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012
Table 5 Estimation of t critical value Quantitative Proton-Nuclear Magnetic
Number of replicates a n t critical value Resonance Spectrometry (1H NMR) for the
2 0.05 1 12.706 Determination of Acetylated Polysaccharides,
Glucose, and Maltodextrin in Aloe Vera Leaf
3 0.05 2 4.303
Juice
4 0.05 3 3.182 The following 1H quantitative NMR (qNMR) method
was developed and validated by Process NMR Associates
strength aloe vera leaf juice; (Danbury, CT), and a similar 1H-NMR approach has
Ct = total aloin concentration in the sample vial from been subjected to independent validation by Spectral
Equation 6, µg/mL; Service (Köln, Germany), Unigen Inc. (Seattle, WA), and
D = dilution factor if applicable; the Department of Chemistry, Saint Martin’s University
Ds = dilution factor, based on the “strength” of the product, (Lacey, WA) (Jiao et al. 2010). The method can be used
estimated by the following equation: for the direct detection and quantitation of the primary
components of interest in aloe vera leaf juice products
Equation 11 and raw materials for compliance with IASC certification
Ds = 1 / x requirements, specifically, for determination of the content
of acetylated polysaccharides, the presence of glucose and
where: malic acid, and the presence and content of maltodextrin
x = “strength” of the liquid concentrate stated in the (see Table 1). Additionally, for meeting quality control
documentation accompanying the material. specifications beyond IASC requirements, the presence
For replicate samples calculate the mean and standard and content of the following groups of compounds can be
deviation/error. Report the total concentration of aloin determined: degradation products (e.g., lactic acid, succinic
utilizing a 95% confidence interval as per the following acid, fumaric acid, acetic acid, formic acid, and ethanol),
formula: preservatives (e.g., potassium sorbate, sodium benzoate, and
citric acid/citrate), and other atypical impurities, additives,
Equation 12
or adulterants (e.g., methanol, glycine, glycerol, sucrose,
maltodextrin, propylene glycol, ethanol). The method
provides advantages over separation-based test methods in
where: that it is rapid, allows for specific recognition of molecular
= mean of replicates of the sample, calculated by the chemistry, requires minimal sample preparation, and is
following equation: quantitative.
The method describes a common internal standard
Equation 13 qNMR methodology that does not require additional
equipment or advanced automation software. There are
other quantitative NMR methods that utilize internal,
calibrated electronic reference signals, as well as the use
where: of multiple standard calibration solutions, that allow direct
n = number of replicates; calculation of the components present in the sample
= t critical value, taken from Table 5; utilizing specialized software automation and spectral
deconvolution algorithms.
= standard error of the mean, calculated by the The method is applicable to a large number of different
following equation: aloe vera raw materials and products, including liquid and
dried juices. In aloe vera finished products the method is
Equation 14
only applicable when the observable aloe vera constituents
are present at a high enough concentration and are not
obscured by additional product ingredients with signals in
where: overlapping areas.
s = standard deviation; Fresh aloe vera leaf juice, produced by processing
n = number of replicates. the entire leaf, contains glucose, malic acid, acetylated
Representative chromatogram of aloins A and B standards polysaccharides, along with citric acid cycle components
are shown in Figure 2. Representative chromatogram of aloe such as citrate, isocitrate, and isocitrate lactone. Some aloe
vera leaf juice is shown in Figure 3. vera leaf juice products and raw materials may also contain
high levels of lactic acid and acetic acid due to malolactic
bacterial fermentation, hydrolysis, or thermal degradation of
the material during production and/or storage. Finished aloe
vera products often contain additives such as preservatives
and flavorants. This method can be readily adapted to

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 33

allow analysis of any or all of these constituents. According
to IASC standards, all aloe vera leaf juice raw materials
should contain not less than 5% dry weight of the acetylated
polysaccharides.
Freeze-drying procedures may lead to the underestima-
tion (or even non-observation) of some of the compounds.
The freeze-drying process can also remove acetic acid,
ethanol, methanol, sorbate, benzoate, and formic acid
from the sample. If these components are of interest to the
manufacturer or marketer of the products being analyzed
then the NMR analysis should be performed on the sample
without freeze-drying (Figure 6). The NMR processing and
final calculations for liquid aloe vera leaf juice samples are
identical to those performed on aloe vera leaf juice powders
and freeze-dried samples. The calculated concentration val-
ues in liquid samples will be much less than the dry weight
values suggested by IASC, as the majority component of the
sample will be water. Weight values will be 10-200 times
less, as the dry matter is typically 0.5%-10% dry weight of
the sample, depending on the concentration of the product.
1
H NMR spectroscopy is not suitable for the observa-
tion of chemical components that are in the ppm (mg/g)
concentration range. In the case of aloe vera chemistry,
NMR is not applicable to the analysis of aloins A and B,
which are expected to be present at less than 10 ppm in
IASC-compliant single-strength aloe vera raw materials and
finished products intended for oral consumption. As only
proton chemistry is observed, it is also not possible to acquire
information on elemental concentrations, such as calcium
and magnesium concentrations.
Note: The NMR chemical shift scale is a normalization method used to
allow direct comparison of NMR spectra obtained at different magnetic field
strengths. Thus, NMR spectra are presented with a scale, in ppm, which is
derived by dividing the absolute frequencies in the spectrum by the resonance
frequency of the observed nucleus (in MHz) on the spectrometer used to
obtain it (ppm = Hz/MHz). The ppm scale is not related to the concentration
of the components being analyzed. It is also important to remember that a
NMR spectrum is not a chromatogram and each peak is not an individual
component molecule. Rather, each of the component molecules gives rise to
multiple specific peaks (singlets, doublets, triplets) at well defined chemical
shifts that correspond to the particular chemical functionality of the protons
(hydrogens) in the sample—whether they are CH, CH2, CH3 and what
functional group they are adjacent to or a part of (aromatics, alkenes, alcohols,
acids, aldehydes, etc.). The area under the NMR peaks is proportional to the
number of protons in that functional group, and knowledge of the sample
chemistry allows the molar ratios of chemical components to be determined.
With knowledge of component molecular weights and the use of appropriate
internal or external standards, it is possible to calculate the concentration of
each component that is identified and properly integrated.
Sample Preparation
Liquid Juice Samples and Aloe Vera-Containing
Commercial Products
Dissolve 150-200 mg of liquid aloe vera juice sample and
5-10  mg of the internal standard (nicotinamide) in ~0.7
mL deuterium oxide (D2O) and transfer into a 5-mm NMR
tube.
Freeze-Dried Juice Samples or Commercially Dried Juice
Products
Dissolve 20-50 mg of dried aloe vera leaf or inner leaf juice
powder and 5-10 mg of the internal standard (nicotinamide)
34 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012
in ~0.7 mL of D2O and transfer into a 5-mm NMR tube.
Note: The exact amount of sample or standard is not important, but weights
must be recorded to the nearest 0.1 mg. Volume of solvent is also not critical
as the final result will be calculated in terms of % weight and does not require
a volume to be used as is required for mg/mL calculations.
Reagents
NMR solvents
D2O (99.9% D-atom) + 0.01mg/ml 4,4-Dimethyl-4-
silapentane-1-sulfonic acid (DSS) (0.7 mL) (e.g., Cambridge
Isotope Laboratories, Andover, MA).
DCl (20% in D2O, 99.5% D-atom) (e.g., Cambridge Isotope
Laboratories, Andover, MA).
Note: Equivalent deuterated solvents from other manufacturers can be used.
qNMR internal standard
Nicotinamide (> 99.5% purity).
Note: Some automated approaches (not described here) require external
standards of glucose, malic acid, lactic acid, and acetic acid, as well as a
standard acetylated polysaccharide solution (e.g., Immuno-10, Unigen,
Seattle, WA). All small molecule components can be obtained from
commercial chemical companies at purity of > 98%.
Equipment
NMR spectrometer
Varian Mercury-300MVX with 1H-19F/15N-31P 5-mm PFG
AutoX DB Probe or 5-mm H/F/P/C 4-nucleus probe.
Operating with Varian VNMR-6.1C software.
Equivalent NMR systems and software include those
from the following manufacturers: Agilent/Varian (VNMR or
VNMRJ software), Bruker (Topspin software), JEOL (Delta
software). The necessary requirements are 1H Resonance
Frequency of 300-500 MHz and a functional 1H probe.
Examples of third party commercial and non-commercial
NMR software capable of processing spectral data acquired
on commercial NMR spectrometers (as above) include
ACD/NMR Processor (ACD/Labs), MNOVA (Mestrelab
Research), SpinWorks (freeware), Chenomx NMR Suite
(Chenomx).
Weighing equipment
Calibrated weighing balance capable of measuring
accurately to 0.1 mg.
Freeze dryer
Virtis BTK Benchtop “K” Manifold (SP Industries) or
equivalent.
Analytical Conditions
The typical NMR instrument parameters are shown in
Table 6. There is some variation of these parameters brought
about by differences in field strength and the sweep width
variations. All experiments must be optimally shimmed and
the acceptance criteria for acceptable spectral performance is
based on the quality of the nicotinamide standard resonance
located at 7.65 ppm which should optimally be a well
resolved, symmetric, 4 peak multiplet. The water resonance
set to 4.8 ppm is utilized as the chemical shift standard in
non-acidified samples. Preferentially internal chemical shift
standards readily available in NMR deuterated solvents DSS
or 3-(trimethylsilyl)-2,2’,3,3’-tetradeuteropropionic acid
(TMSP-d4) can also be utilized as the reference for 0 ppm.
Table 6 Typical NMR instrument parameters Table 7 1H NMR method limits of detection (LOD) and quantitation
(LOQ) for some of the constituents naturally present in aloe vera
Acquisition Time 3-8 seconds leaf juice
Relaxation (Recycle) Delay 2-6 seconds Signal-to-Noise Signal-to-Noise ratio
Frequency, MHz 300-500 MHz Substance ratio (S/N) > 3 (S/N) > 10

Nucleus 1
H LOD, mg/mL LOQ, mg/mL

Number of Pulse Accumulations* 16-256 Acetylated

mannan < 0.05 < 0.1
Original FID Points 16384-84000
Glucose < 0.05 < 0.05
Zero-filled Points 32768-262144
Malic acid < 0.05 < 0.05
Pulse sequence Single pulse
Lactic acid < 0.005 < 0.005
Solvent D2O
Acetic acid < 0.001 < 0.005
Sweep width, ppm 16

Temperature Ambient (25 ºC)

naturally present in aloe vera leaf juice are presented in
Line Broadening 0.35 Hz Table 8. Additionally, the chemical shifts, molar conversion
Steady State Pulses 8 factors, and peak descriptions for compounds indicative
of degradation of aloe vera acetylated polysaccharides, as
Pre-Acquisition Delay 60 seconds well as those for compounds that may be present in aloe
* Number of transients depends on the component concentration present vera juice products and raw materials as preservatives and
in the sample being analyzed. Signal-to-noise (S/N) must be high (>10:1 additives, are shown given in Table 9.
for the smallest component signal to be quanitified, >3:1 on smallest
Detection of Glucose
component to be detected). The analyst must decide the appropriate
Glucose has two natural anomers resulting in two signals
number of transients to obtain adequate S/N.
at 4.6 ppm and 5.2 ppm, respectively, for the C-1 (α and β)
protons (see Figure 4).
The DSS, TMSP, or component line-shapes should also be
Detection of Maltodextrin
utilized to validate the lineshape and thermal stability of
Maltodextrin may be added to powdered aloe vera leaf
the acquisition. Other resonances in the sample that can be
juice during spray-drying. If content of maltodextrin is not
used for confirmation of line shape are glucose (doublet at
correctly stated on the documentation accompanying the
5.2 ppm), lactic acid (if present, 1.35 ppm). The analysis can
product, it is considered an adulterant. A single, relatively
be performed by an appropriately trained technician under
broad 1H NMR signal at 5.4 ppm indicates the presence
the supervision of a qualified NMR spectroscopist, however,
of maltodextrin. If the signal at 5.4 ppm is narrow and
the data processing, quantitative analysis, and decisions on
shows the structure of a doublet then the signal is due
experimental approaches (acidification of sample followed
to the anomeric proton signal of sucrose rather than
by second NMR analysis) should be performed by a
maltodextrin. Figure 7 shows an example of aloe vera leaf
qualified NMR spectroscopist.
juice powdered concentrate that contains 75% maltodextrin.
Limits For quantitation of maltodextrin, see “Quantitation” below.
The limit of detection (LOD) and limit of quantitation
Quantitation
(LOQ) values for some of the aloe vera leaf juice components
After the component signals have been properly assigned
calculated for this method can be seen in Table 7. The
and identified (see Tables 8 and 9), the component signals
LOD/LOQ values can vary based on the spectrometer
are carefully integrated and the integral values transferred
field strength, NMR probe type and configuration, and
into spreadsheets or utilized in NMR software macros and
post-processing procedures such as apodization. For full
automation routines. Some advanced software packages
description of typical LOD, LOQ, linearity, robustness,
might also allow automatic identification and integration
accuracy and reproducibility results of the method, see Jiao
of the signals of interest, or completely deconvolute and
et al. (2010).
quantify the components based on spectral deconvolution
Detection using pure component spectra as a basis set.
1
H-NMR spectra are collected with analytical parameters
Quantitation of Acetylated Polysaccharides
equal or close to those shown in Table 6. The spectra are
The multiple NMR peaks associated with acetylation groups
processed with manual or automatic phase correction,
are found between 2.0 and 2.3 ppm and these have been
baseline correction, and manual integration of resonance
chosen as the characteristic peaks for acetylated mannose
signals. Characteristic chemical shift values, molar
residues in aloe vera polysaccharides. Quantitation of
conversion factors, and peak descriptions, for compounds
acetylated polysaccharides by 1H NMR, where the repeat
American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 35
Table 8 Characteristic chemical shift values, peak multiplicity, Equation 3
protonated carbon type, and molar conversion factors
CAP = ((WNic * IAcMann * NNic * MWAcMann) / (INic * NAcMann * MWNic)
(N-parameter) used for detection and quantitation of the major
natural components of aloe vera leaf juice + (WNic * IAcMann * NNic * MWMann * (0.22 / 0.78)) / (INic * NAcMann *
MWNic)
Substance Signal type and Chemical shift,
+ (WNic * IAcMann * NNic * MWGl * (0.16 / 0.84)) / (INic * NAcMann *
N-parameter* ppm
MWNic))
Acetylated Broad group of CH3 2.0-2.3 * (1 / Wsample) * 100%
mannan singlets (N=3)

Isocitric acid CH, doublet (N=1) 4.25 where:

CAP = content of acetylated polysaccharides in the
Malic acid CH, 4-peak multiplet 4.45
sample, % weight;
(N=1)
WNic = weight of added internal standard (nicotinamide),
a-Glucose CH, doublet (N=1) 4.6 mg;
IAcMann = integration area of acetylation methyls (multiple
b-Glucose CH, doublet (N=1) 5.2
peaks in 2.0-2.3 ppm region);
Isocitric CH, doublet (N=1) 5.05 NNic = molar conversion factor (see Table 8) for
lactone nicotinamide = 4;
* N-parameter value refers to the number of protons in the functional group
MWAcMann = molar weight of acetylated mannosyl residue =
by which the integrated signal intensity must be divided in order to obtain 204.2 g/mol;
molar ratios that can then be converted to % weight values. INic = sum of integration areas of the 4 aromatic CH
peaks of the nicotinamide standard;
NAcMann = molar conversion factor for acetylmannosyl = 3;
units of the polymer yield superimposed signals in the MWNic = molar weight of nicotinamide standard
NMR spectrum is accomplished by multiplication of the = 122.1 g/mol;
molar integrated signal values in the 2.0-2.3 ppm region MWMann = molar weight of non-acetylated mannosyl residue
by the molar weight of the average monomer unit. In the = 162.2 g/mol;
case of aloe vera acetylated polysaccharides, it has been MWGl = molar weight of glucosyl and galactosyl residues
demonstrated that the acetylation of the mannose monomer = 162.2 g/mol;
units is at 78% (Manna and McAnalley 1993) and that Wsample = weight of sample, mg.
mannose represents 84% of the polysaccharide backbone
with the remainder being composed of glucose, galactose, Substituting the constant parameters with numerical values,
and a few other saccharides (Chow et al. 2005). The the following formula for estimating the concentration of
acetylation content and the presence of other saccharides acetylated polysaccharides (CAP, % weight) is obtained:
must be taken into account so as not to underestimate the Equation 4
acetylated polysaccharides content. CAP = 3.06 * (WNic * IAcMann) / (INic * Wsample) * 100%
The molar weight (MW) of an acetylated mannosyl
monomer is calculated by subtracting the molar weight where:
of two water molecules: one removed upon condensation CAP = content of acetylated polysaccharides in the sample,
of two mannose monosaccharides, and another upon the % weight;
condensation of acetate with mannosyl monomer. Thus: WNic = weight of added internal standard (nicotinamide),
Equation 1 mg;
MWmannosyl = MWmannose – MWwater = 180.2 g/mol – 18 g/mol IAcMann = integration area of acetylation methyls (multiple
= 162.2 g/mol peaks in 2.0-2.3 ppm region);
INic = sum of integration areas of the 4 aromatic CH peaks
Equation 2 of the nicotinamide standard;
MWacetylmannosyl = MWmannosyl + MWacetate – MWwater Wsample = weight of sample, mg.
= 162.2 g/mol + 60 g/mol – 18 g/mol
= 204.2 g/mol Quantitation of Maltodextrin
According to IASC criteria, if maltodextrin is added to aloe
Now, taking into account the 0.78/1 acetyl/mannosyl ratio vera leaf juice during manufacturing, its content should be
(Manna and McAnalley 1993) as well as the reported accurately declared; otherwise, it is considered an adulterant.
presence of 16% non-mannosyl saccharides (mostly glucose Figure 7 shows an example of aloe vera leaf juice powdered
and galactose) in the aloe vera acetylated polysaccharides concentrate that contains 75% weight of maltodextrin and
(Chow et al. 2005), we can calculate the concentration 25% weight of leaf juice powder. The peak at 5.4 ppm can
of the acetylated polysaccharides (CAP) by the following be used to determine the quantity of maltodextrin present.
equation (Equation 3): Care must be taken to distinguish between the broad single
resonance of maltodextrin and the doublet that arises at the

36 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012

Table 9 Chemical shift values, peak descriptions, and molar conversion factors that can be used for detection and quantitation of aloe vera
leaf juice preservatives, additives, and degradation products

Compound Type of compound Signal Chemical shift, ppm

Propylene glycol Additive CH3, doublet (N=3) 1.1

Ethanol Degradation product or additive CH3, triplet (N=3) 1.15

Lactic acid Degradation product CH3, doublet (N=3) 1.33

Potassium sorbate Preservative CH3, doublet (N=3) 1.82

Acetic acid Degradation product CH3, singlet (N=3) 1.96

Pyruvic acid Degradation product CH3, singlet (N=3) 2.35

Citric acid Naturally present or added as pH regulator and 2 x CH2, multiplet (N=4) 2.5-3.0
preservative

Succinic acid Degradation product 2 x CH2, singlet (N=4) 2.6

Glycerol Additive CH2 and CH, multiplet 3.5

Glycine Additive CH2, singlet (N=2) 3.51

Sucrose Additive CH, doublet (N=1) 5.4

Fumaric acid Degradation product 2 x CH, singlet (N=2) 6.5

Sodium benzoate Preservative 2 x CH, doublet (N=2) 7.95

Formic acid Degradation product CH, singlet (N=1) 8.2-8.3

same position from sucrose. The formula for quantitation of IMDX = integration area of anomeric proton resonance of
maltodextrin is shown in the Equation 5. maltodextrin (5.4 ppm);
Equation 5
INic = sum of integration areas of the 4 aromatic CH peaks
CMDX = (WNic * IMDX * NNic * MWMDX monomer)
of the nicotinamide standard;
/ (INic * NMDX * MWNic * Wsample) * 100%
Wsample = weight of sample, mg.

Additional Components (optional analysis)

where: Multiple components are commonly present in aloe vera
CMDX = content of maltodextrin in the sample, % leaf juices. These include malic acid, glucose, products
weight; of degradation of the acetylated polysaccharides and small
WNic = weight of added internal standard, mg; molecule components, and various additives. Although
IMDX = integration area of anomeric proton resonance unnecessary for IASC certification, information about such
of maltodextrin (5.4 ppm); substances may be useful for assessing the quality of
NNic = molar conversion factor for nicotinamide = 4; the material. Use of 1H NMR allows detection and, if
MWMDX monomer = molar weight of maltodextrin monomer needed, quantitation of the levels of these compounds
= MWglucose – MWwater = 180.2 g/mol – 18 g/mol without additional runs or standards. 1H NMR will also
= 162.2 g/mol; allow the direct observation and identification of unknown
INic = sum of integration areas of the 4 aromatic CH contaminants that might appear in aloe vera raw materials
peaks of the nicotinamide standard; and products.
NMDX = molar conversion factor for maltodextrin = 1;
MWNic = molar weight of nicotinamide = 122.1 g/mol; The general formula to calculate the concentration (%
Wsample = weight of sample, mg. weight) of any component is:
Equation 7
After substitution with constant numerical values, the
CX = (WNic * IX * NNic * MWX) / (INic * NX * MWNic * Wsample)
following formula is obtained:
* 100%
Equation 6
CMDX = 5.32 * (WNic * IMDX ) / (INic * Wsample) * 100%
where:
CX = concentration of the component, % weight;
where:
CMDX = content of maltodextrin in the sample, % weight; WNic = weight of added internal standard (nicotinamide),
WNic = weight of added internal standard, mg;
mg;

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 37


Figure 4 Typical 1H-NMR spectrum of freeze-dried aloe vera leaf
juice
Discussion
The presence of molecular components of interest in aloe vera
leaf juice can be established from observing this spectrum. Peaks
representing a- and b-glucose are observed at 4.6 ppm and
5.2 ppm, respectively. The group of peaks associated with the
acetylated CH3 groups in the aloe vera acetylated polysaccharides
is located in the 2.0–2.3 ppm region. The presence of multiple
degradation products (pyruvic acid, acetic acid, lactic acid,
succinic acid, formic acid, and ethanol) and/or additives (sorbate
IX = integration area of the unique proton resonance
from the component spectrum;
NNic = molar conversion factor for nicotinamide = 4
(the number of the aromatic CH groups in the
molecule);
MWX = molar weight of the component, g/mol;
INic = sum of integration areas of 4 aromatic CH peaks of
the nicotinamide standard;
NX = molar conversion factor for the compound (equal
to the number of protons in the resonance group/
groups): CH = 1, CH2 = 2, CH3 = 3;
MWNic = molar weight of nicotinamide = 122.1 g/mol;
Wsample = weight of sample, mg.
The formulas for estimation of several additional constituents
of interest in aloe vera leaf juice are provided further.
38 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012
and citric acid) can be established by the observation of the
indicated peaks. The peaks in the 3.2–4.3 ppm area represent
polysaccharide and glucose sugar signals. In addition, several
peaks associated with isocitrate and isocitrate lactone are
observed and quantified, indicating that this is a leaf juice (i.e.,
not inner leaf juice) product. As mentioned previously, the NMR
spectroscopist responsible for acquisition and analysis of the data
must make a decision as to whether acidification of the sample is
required in order to shift the isocitrate CH resonance (indicated
by ‘x’) into a region of the spectrum where it can be quantitatively
integrated (see Figure 7).
Malic Acid:
The malic acid CH signal at 4.3 ppm is usually present as a
multiplet but is often broadened due to chelation effects. In
some cases there is considerable complexation of malic acid
with other components in the juices. This can lead to broad
resonances for malic acid that are more difficult to properly
integrate and quantify, particularly in powdered aloe vera leaf
juice samples. The addition of a mineral acid (DCl) breaks
up the malic acid complexes and allows proper integration
of the malic acid CH signals (Figure 7). As stated previously,
the pH of the final solution is not important if the analysis
is to be performed by an analyst. However, the pH must be
known and controlled if automated spectral deconvolution
methods are to be used. Provided that the molar weight of
malic acid is 134.1 g/mol, and the resonance peak used for
estimation is CH (N = 1), the calculation is the following
(Equation 8):
Figure 5 1H-NMR spectrum of a freeze-dried 5x aloe vera leaf juice
Discussion
The nicotinamide standard yields peaks at 7.7 ppm and in the 8.2-
9.0 ppm region. In leaf juice samples, it is often found that there
has been thermal or enzymatic degradation of the acetylated
polysaccharides with the result of producing a different shape
distribution in the three acetylated mannosyl CH3 resonances (2.0-
2.3 ppm).

Equation 8 WNic = weight of added internal standard (nicotinamide),

CMA = 4.39 * (WNic * IMA) / (INic * Wsample) * 100% mg;
where: Ia = integration area of the CH proton resonance of
CMA = concentration of malic acid in the sample, % a-glucose (5.2 ppm);
weight; Ib = integration area of the CH proton resonance of
WNic = weight of added internal standard (nicotinamide), b-glucose (4.6 ppm);
mg; INic = sum of integration areas of the 4 aromatic CH
IMA = integration area of the CH proton resonance of peaks of the nicotinamide standard;
malic acid (4.3 ppm); Wsample = weight of sample, mg.
INic = sum of integration areas of the 4 aromatic CH Isocitrate:
peaks of the nicotinamide standard; The instructions for isocitrate quantitation are provided in
Wsample = weight of sample, mg.
the Aloe Vera Inner Leaf Juice monograph.
Glucose: Isocitrate Lactone:
The anomeric proton signals of both the a (doublet at The CH resonance (doublet at 5.05 ppm) of the isocitrate
5.2 ppm) and b (doublet at 4.6 ppm) anomers of glucose lactone molecule (176.0 g/mol) is utilized for quantitation.
are used to quantify the glucose content of aloe vera leaf The final formula is the following:
juice. The molar weight of glucose is 180.2 g/mol, and the
resonance groups used for quantitation are CH (N = 1). The Equation 10
calculation is the following (Equation 9): CICL = 5.77 * (WNic * IICL) / (INic * Wsample) * 100%

Equation 9
where:
CGlu = 5.90 * (WNic * (Ia + Ib)) / (INic * Wsample) * 100%
CICL = concentration of isocitrate lactone in the sample,
% weight;
where: WNic = weight of added internal standard (nicotinamide),
CGlu = concentration of glucose in the sample, % weight;
mg;

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 39


Figure 6 1H-NMR spectrum of a 5x aloe vera leaf juice analyzed without freeze-drying
Discussion
This spectrum shows a 5x aloe vera leaf juice with benzoate and
sorbate added as preservatives. All signals typically observed in
freeze-dried 5x leaf juice are observed, but at a lower intensity.
A large H2O resonance is observed at 4.8 ppm. The spectrum
shows some degradation products, such as acetic acid (singlet
at 1.98 ppm) and lactic acid (doublet at 1.3 ppm), and signs of
IICL = integration area of the CH proton resonance of
isocitrate lactone (5.05 ppm);
INic = sum of integration areas of the 4 aromatic CH
peaks of the nicotinamide standard;
Wsample = weight of sample, mg.
Citrate:
Citrate (192.0 g/mol) CH2 resonances appear in the 2.5-3.0
ppm region and are overlapped by the CH2 resonances of
malic acid, isocitrate, and isocitrate lactone. These latter
components can be quantified from their CH resonances
found in the 4.25-5.05 ppm region. Thus, once the integrated
values for the CH protons of malic acid (IMA), isocitric acid
(IICA), and isocitrate lactone (IICL) have been obtained, it is
possible to obtain the molar integration value of citric acid
(ICA / NCA) by subtracting the intensity of these CH carbons
(multiplied by 2 to adjust for the CH2 signal intensities
being subtracted for these molecules) from the CH2 region,
and then dividing by the four citric acid protons (NCA) that
are present in that region. The final calculation is shown in
Equation 11.
40 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012
fermentation (ethanol CH3 triplet at 1.15 ppm). The two doublet
signatures observed between 2.8 and 3.0 ppm are indicative of the
presence of isocitrate. Isocitrate is quantified only if the material
is labeled as “inner leaf juice” (see the Aloe Vera Inner Leaf Juice
monograph).
Equation 11
CCA = 1.57 * (WNic * ICA) / (INic * Wsample) * 100%
where:
CCA = concentration of citric acid in the sample, % weight;
WNic = weight of added internal standard (nicotinamide), mg;
ICA = I2.5-3.0 – 2 * (IICL + IICA + IMA)
where:
I2.5-3.0 = total integration area of the 2.5-3.0 ppm region;
INic = sum of integration areas of the 4 aromatic CH peaks
of the nicotinamide standard;
Wsample = weight of sample, mg.
Degradation Products:
The presence of degradation products can be used to assess
the quality of the material and give indications of sample
processing and storage conditions. Chemical shift values,
peak descriptions, and molar conversion factors for typical
degradation products of aloe vera acetylated polysaccharides
can be found in Table 9.

 
Figure 7 1H-NMR spectra of a commercial aloe vera leaf juice powder containing 75% dry weight maltodextrin, before and after acidification
with 1 drop of 20% DCl in D2O
Discussion of 20% DCl in D2O allows the proper quantitation of the isocitrate
The region around 2.8-3.0 shows the presence of isocitrate in the by shifting the pH sensitive resonance to 4.55 ppm where it can
non-acidified sample (lower spectrum). Acidification with 1 drop be properly integrated without overlapping signals from other
component molecules.

Preservatives and Additives:

Chemical shift, peak descriptions and molar conversion
factors for common preservatives, flavorants, and formulation
additives used in aloe vera leaf juice products are provided
in Table 9.

Unexpected Components:
The 1H-NMR method can also identify and quantify the
presence of unexpected compounds which might prove
harmful if present at high concentration. One example of
this is the observation of methanol (CH3, singlet, at 3.3 ppm)
in some aloe vera leaf juice materials.

Limit Tests
Solids Content: Not less than 1.0% in single-strength (1×)
juice, determined by drying a 10-gram
sample of the liquid at 105 ºC for 24 hours
(Wang and Strong 1995).

American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012 41

  
Figure 8 1H-NMR spectrum of a commercial, freeze-dried 100x aloe vera leaf juice powder
Discussion degradation products as well as the presence of isocitrate. In
This spectrum of a commercial powder produced by freeze-drying order to obtain quantitative isocitrate results, this sample would
aloe vera leaf juice shows the typical CH3 peak distribution of the have to be acidified to shift the isocitrate CH resonance into an
acetylated polysaccharides. It also shows the presence of acetic area of the spectrum where it is not overlapped.
acid (1.98 ppm), lactic acid (1.33 ppm), and formic acid (8.4 ppm) be properly integrated without overlapping signals from other
component molecules.

42 American Herbal Pharmacopoeia® • Aloe Vera Leaf Juice • 2012

Aloe Vera Inner
Leaf Juice
Aloe vera (L.) Burm. f.
Nomenclature
Pharmacopoeial Definition
Aloe vera inner leaf juice consists of the liquid expressed
from the clear inner layer of the leaves of Aloe vera (L.)
Burm. f. or the dry powdered concentrate of such liquid.
Inner leaf juice contains not less than 5% dry weight of
acetylated polysaccharides and not more than 5% dry weight
of isocitric acid, determined by proton nuclear magnetic
resonance spectrometry (1H NMR).
Identification
Macroscopic Identification
Aloe vera inner leaf juice is a viscous, cloudy to transparent
liquid.
Organoleptic Characterization
Liquid (single-strength)
Color: Clear to caramel-colored.
Aroma: Odorless to mildly vegetative.
Taste: Tasteless to slightly bitter.
Flakes and powder
Color: Beige.
Aroma: Odorless to mildly vegetative.
Taste: Tasteless to slightly bitter.
Commercial Sources
and Handling
Aloe vera inner leaf juice is produced from the clear
inner leaf, separated from the rind in the process called
“filleting.” During filleting, whether manual or mechanical,
care must be taken not to introduce excessive amounts of
phenolic constituents located in the vascular layer of the
leaf. If contamination with these compounds does occur,
decolorization may be employed to reduce the levels to the
10 ppm limit in the single-strength raw material or finished
product (Table 1). Aloe vera inner leaf juice is prone to
enzymatic, thermal, and bacterial degradation. For a more
thorough discussion of production of aloe vera inner leaf
juice, see the Aloe Vera Leaf monograph.
American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012
Constituents
The solid fraction of the juice produced from aloe
vera inner leaf consists predominantly of carbohydrates
(acetylated polysaccharides, free glucose), organic acids
(predominantly malic acid) and mineral ions (Femenia
et al. 1999; Waller et al. 2004). A review of a limited
number of commercial aloe vera “gel” products suggests
that polysaccharide concentration varies widely, with a
large percentage of products containing lower than 10%
acetylated polysaccharides and some containing as low as
1% (Bozzi et al. 2007). “Filleting” the leaves prior to making
the juice removes most of the phenolic compounds from
the final product. To further reduce the phenolic content,
if needed, decolorization can be used. The characteristic
constituent profile of aloe vera inner leaf juice is shown in
Table 2.
A na ly t i ca l
Maltodextrin Assay
Proceed as directed in the Aloe Vera Leaf Juice monograph.
High Performance Liquid Chromatography
(HPLC) for the Quantification of Aloins A and
B in Aloe Vera Inner Leaf Juice
The International Aloe Science Council requires not more
than 10 ppm of total aloins in single-strength (1×) aloe vera
inner leaf juice raw materials and finished products. For
the determination of quantity of aloins A and B in aloe vera
inner leaf juice products, the same HPLC procedure as
described in the Aloe Vera Leaf Juice monograph should
be used.
Sample Preparation, Standards Preparation,
Instrumentation, Reagents, Procedure and System
Suitability
Proceed as directed in the Aloe Vera Leaf Juice monograph.
Calculations
Obtain the integrated area (I) values of aloin A in the
calibration standards and aloins A and B in the analyzed
samples. Construct a plot of concentration (µg/mL) of the
aloin A calibration standard (x-axis) against the individual
peak area (y-axis). Calculate the slope (m), intercept (b) and
correlation coefficient (R2) of the best-fit line.
43
Table 1 IASC certification requirements for aloe vera inner leaf Table 2 Typical constituent profile of aloe vera inner leaf juice as
juice determined by 1H-NMR

Compound Certification Requirement Component Content*(% dry weight)

Acetylated Acetylated mannan 8.98

≥ 5% dry weight
polysaccharides Glucose 12.89
Glucose Present Malic acid 23.16
≤ 10 ppm in single-strength inner leaf Lactic acid 0.06
juice, analyzed by HPLC or other fit-
Aloin Citric acid 5.47
for-purpose methodology approved
by the IASC Pyruvic acid 0.04

Must be listed on label and analysis Sorbate 0.73

Maltodextrin
must meet label claims. Benzoate 0.53
≥ 0.5% in single-strength inner leaf Isocitrate 0.00
Solids
juice
Isocitrate lactone 0.00
Ash ≤ 40%
Acetic acid 0.27
Isocitrate ≤ 5% dry weight Succinic acid 0.00
Formic acid 0.00
The concentration of aloin A or aloin B in the prepared Fumaric acid 0.00
sample is calculated by the following equation:
Ethanol 0.15
Equation 1
Maltodextrin 0.00
Cv = (Ialoin A/B – b) / m *
Content determined in a freeze-dried sample.

where:
Cv = concentration of aloin A or B in the sample vial, µg/
mL;
Ialoin A/B = integrated area of the aloin A or B peak in the
sample;
b = intercept of the calibration curve;
m = slope of the correlation curve.
Determine the concentration of total aloin (Ct, µg/mL) in
the sample vial by adding individual aloin concentrations:
Equation 2
Ct = Cv of aloin A + Cv of aloin B
accompanying the material, such as certificate of analysis
(COA). Pure aloe vera inner leaf juice powder is usually
designated as having 200× strength, based on the standard
value of the solids content in aloe vera inner leaf juice of
5 mg/mL (0.5% w/vol). Lower strengths indicate that other
agents (e.g., carriers) have been added during the drying
process. The following formula can be used to determine
the total aloin concentration in single-strength preparations:
Equation 4
C1x = Cs * Ds * S

For powdered material, the concentration of aloin A and where:

B in the original sample is calculated by the following
equation:
Equation 3
Cs = Ct * V1 * D / Ms
where:
Cs = concentration of total aloin in the original sample, µg/
mg;
Ct = concentration of total aloin A and B in the sample vial,
µg/mL, determined in the Equation 2 above;
V1 = volume of the prepared sample, mL (here 1 mL);
D = dilution factor if applicable;
Ms = mass of original sample, mg.
To determine total aloin concentration in single-strength
inner leaf juice, first obtain the “strength” value of the
juice, typically stated on the label or in the documentation
44 American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012
C1x = concentration of total aloin, ppm or µg/mL, in single-
strength aloe vera inner leaf juice;
Cs = total aloin concentration in the sample, determined in
the Equation 3 above, µg/mg;
Ds = dilution factor, based on the “strength” of the product,
estimated by the following equation:
Equation 5
Ds = 200 / x
where:
x = “strength” of the powdered concentrate stated in
the documentation accompanying the material;
S = standard concentration of juice solids in single-strength
aloe vera leaf juice = 5 mg/mL.
Aloe vera inner leaf juice can also be marketed as liquid
concentrates, e.g., 5×, etc. For liquid material, first obtain
the “strength” value of the liquid and determine the total
Table 3 Estimation of t critical value Quantitative Proton-Nuclear Magnetic
Number of replicates a n t critical value Resonance Spectrometry (1H NMR) for the
2 0.05 1 12.706
Identification of Acetylated Polysaccharides,
Glucose, Maltodextrin, and Isocitrate in Aloe
3 0.05 2 4.303
Vera Inner Leaf Juice
4 0.05 3 3.182 The 1H-NMR method can be used for the direct detection
and quantitation of the primary components of interest in
content of aloins A and B in single-strength juice by the aloe vera inner leaf juice products for compliance with IASC
following equation: certification requirements, specifically, for determination of
the content of acetylated polysaccharides, the presence
Equation 6
of glucose and malic acid, the presence and content of
C1x = Ct * D * Ds maltodextrin, and the content of isocitrate (see Table 1).
Additionally, the presence and content of the following
compounds can be determined: degradation products (e.g.,
where:
lactic acid, succinic acid, fumaric acid, acetic acid, formic
C1x = total concentration of aloins, ppm or µg/mL, in single-
acid, and ethanol), preservatives (e.g., potassium sorbate,
strength aloe vera inner leaf juice;
sodium benzoate, and citric acid/citrate), and other atypical
Ct = total aloin concentration in the sample vial from
impurities, additives, or adulterants (e.g., methanol, glycine,
Equation 2, µg/mL;
glycerol, sucrose, maltodextrin, propylene glycol, ethanol).
D = dilution factor if applicable;
There are three main constituents present in fresh
Ds = dilution factor, based on the “strength” of the product,
aloe vera inner leaf juice produced by processing the inner
estimated by the following equation:
gel of the aloe leaf. These are acetylated polysaccharides,
Equation 7 glucose, and malic acid. According to IASC standards, all
Ds = 1 / x aloe vera inner leaf juice raw material should contain not
less than 5% dry weight of the acetylated polysaccharides. In
where: addition, IASC-certified raw materials and products labeled
x = “strength” of the liquid concentrate stated in the as aloe vera inner leaf juice must contain not more than 5%
documentation accompanying the material. dry weight of isocitrate. IASC-certified raw materials and
For replicate samples calculate the mean and standard products with isocitrate levels of more than 5% dry weight
deviation/error. Report the total concentration of aloin are defined as “aloe vera leaf juice,” in accordance with
utilizing a 95% confidence interval as per the following IASC nomenclature.
formula: Sample Preparation, Reagents, Equipment, Analytical
Equation 8 Conditions, Limits, Detection of Glucose and
Maltodextrin
Proceed as directed in the Aloe Vera Leaf Juice monograph.
Quantitation
where:
For quantitation of acetylated polysaccharides, maltodextrin,
= mean of replicates of the sample, calculated by the
and additional optional components, proceed as directed in
following equation:
the Aloe Vera Leaf Juice monograph.
Equation 9
Quantitation of Isocitrate
In aloe vera leaf juice that contains large amounts of
material from the outer rind, the 2.5–3.0 ppm region of
the spectrum is further complicated by the presence of
where: compounds that occur in the green outer part of the aloe
n = number of replicates; leaf. Their presence leads to overlapping of the signals of
= t critical value, taken from Table 3; the CH2 protons of malic acid and citric acid with those of
isocitrate and isocitrate lactone. This means that the 2.5–3.0
= standard error of the mean, calculated by the ppm region cannot be used directly for the quantitation of
following equation: these components. Instead, quantitation is performed in
Equation 10 the region of the spectrum where the CH resonances for
isocitrate are found: a doublet at 4.25 ppm.
The lactic acid CH quartet and isocitrate CH doublet
signals may overlap in the 4.1–4.2 ppm region. This will
where:
not be determined until after the initial 1H-NMR analysis
s = standard deviation;
has been performed and the concentration of lactic acid
n = number of replicates.
ascertained. If it is found that there is an overlap, the isocitrate
American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012 45

Figure 1 Example spectra of freeze-dried inner leaf juices (a)
with and (b) without preservatives
Discussion
The spectra show the predominance of glucose, malic acid,
and acetylated polysaccharides. In both samples it can be seen
that the acetylated polysaccharide is intact and has undergone
little thermal or enzymatic degradation. However, the presence
of lactic acid shows that some malolactic fermentation has
concentration must be calculated from an observation of the
1
H-NMR spectrum after pH adjustment with a single drop
of concentrated DCl (or any other deuterated mineral acid).
The addition of the mineral acid shifts the isocitrate CH
signal into an area of the spectrum where it is free from
interference (~ 4.45 ppm) and can be properly integrated.
The pH of the final solution is not important if the analysis
is to be performed by an analyst. However, the pH must be
known and controlled if automated spectral deconvolution
methods are to be used. Thus, the formula for quantitation
of isocitrate is the following:
Equation 1
CICA = (WNic * IICA * NNic * MWICA) / (INic * NICA * MWNic * Wsample) * 100%
where:
CICA = content of isocitrate in the sample, % weight;
46 American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012
occurred. The presence of sorbate and benzoate preservatives
can be readily identified, and the compounds can be quantified if
wanted. The nicotinamide standard yields peaks at 7.7 ppm and
in the 8.2–9.0 ppm region. No isocitrate or isocitrate lactone are
observed in the 2.9-3.1 and 5.05 ppm region. When integrals are
obtained on the glucose CH and malic acid CH resonances, it is
important to remove the signal intensity from broad underlying
resonances. This can be done by deconvolution techniques or by
dc-corrections after expanding tightly into the peak.
WNic = weight of added nicotinamide internal standard,
mg;
IICA = integration area of CH proton resonance of
isocitrate (doublet at 4.45 ppm (dissolved in D2O
and acidified with DCl), or 4.2 ppm (dissolved in
D2O));
NNic = molar conversion factor for nicotinamide = 4;
MWICA = molar weight of isocitrate = 192.1 g/mol;
INic = sum of integration areas of the 4 aromatic CH
peaks of the nicotinamide standard;
NICA = molar conversion factor for isocitrate = 1;
MWNic = molar weight of nicotinamide standard = 122.1 g/
mol;
Wsample = weight of sample, mg.
Or, after substitution with constant numerical values:
Figure 2 1H-NMR spectrum of a 10x aloe vera inner leaf juice
analyzed without freeze-drying
Discussion
The spectrum shows an example of a 10x inner leaf juice analyzed
without freeze-drying. Sorbate, acetic acid, and ethanol are
partially lyophilized from samples during the freeze-drying process.
If these components are important in the analysis they must be
calculated from the 1H-NMR spectrum of the juice sample that has
not been freeze-dried. All signals typically observed in freeze-dried
10x inner leaf juice are observed, but at a lower intensity. A large
H2O resonance is observed at 4.8 ppm.

Equation 2 Limit Tests

CICA = 6.29 *(WNic * IICA) / (INic * Wsample) * 100%
Solids Content: Not less than 0.5% in single-strength (1×)
juice, determined by drying a 10-gram
where:
sample of the liquid at 105 ºC for 24 hours
CICA = content of isocitrate in the sample, % weight;
(Wang and Strong 1995).
WNic = weight of added nicotinamide internal standard,
mg;
IICA = integration area of CH proton resonance of
isocitrate (doublet at 4.45 ppm (dissolved in D2O
and acidified with DCl), or 4.2 ppm (dissolved in
D2O));
INic = sum of integration areas of the 4 aromatic CH peaks
of the nicotinamide standard;
Wsample = weight of sample, mg.

American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012 47


Figure 3 Examples of a commercial 200x inner leaf juice powder
and a 100x inner leaf juice powder containing 50% maltodextrin
Discussion
Maltodextrin can be readily observed by the presence of a
broad intense resonance at 5.4 ppm along with accompanying
broad resonances at 3.9, 3.8, and 3.6 ppm. If the resonance at
5.4 ppm is a well-resolved doublet then the analysis must be
performed assigning that type of resonance to sucrose. The
48 American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012
acetylated polysaccharides have a broad “three-peak” multiplet
appearance. The integral utilized in the calculations of acetylated
polysaccharide content encompasses all three of the broad
resonances. The small peak observed in the 100x powder at 2.0
ppm is assigned to acetic acid. In thermally degraded samples,
deacetylation of the mannosyl residues leads to a change in
the observed distribution of the three peaks in the acetylated
polysaccharide CH3 resonances, which is accompanied by the
increase in the intensity of the acetic acid CH3 resonance.

Figure 4 1H-NMR spectrum of a commercial 200x aloe vera inner
leaf juice powder showing the effect of acidification of the
sample on the 1H-NMR spectrum
Discussion
In cases when the material or product is labeled as inner leaf juice,
the contents of isocitrate must be quantified. In many samples
the isocitrate CH resonance utilized in the analysis to quantify the
concentration is severely overlapped by sugar and other small
molecules signals (especially the lactic acid CH resonance).
Acidification of the sample with mineral acid (DCl), by adding
American Herbal Pharmacopoeia® • Aloe Vera Inner Leaf Juice • 2012
a single drop of 20% DCl in D2O, shifts the malic acid, citrate,
isocitrate, and isocitrate lactone signals (as well as formic acid,
acetic acid, succinic acid, and the nicotinamide standard signals),
increasing the resolution of the malic acid/citrate/isocitrate CH2
region (2.4-3.0 ppm). Acidification can also cause the malic acid CH
resonance to overlap with the b-glucose resonance. The isocitrate
CH resonance, which is of main interest in this part of the analysis,
is typically found as a discrete doublet not overlapped with any
other signals, as indicated at 4.5 ppm. All other components are
calculated from the spectrum before acidification.
49
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