Medicine Update, 2002, Vol. 10, No.

7 LIVER UPDATE

Reduction of HBsAg with decrease in viral load with

herbal formulation, HD-03/ES: A case study

Dr. KALA SUHAS KULKARNI, Dr. MALLIKARJUN N.DIXIT, Dr.V.G.BHAGWAT,

Dr.ASHWIN KUMAR MERU, Dr.U.V.BABU, Dr.EKTA SAXENA, Dr.S.K.MITRA

R&D Center, The Himalaya Drug Company, Makali, Bangalore

INRODUCTION

The hepatitis B virus (HBV) is globally distributed among humans. The various strains of
HBV are quite species specific.1 Many individuals are affected worldwide, but the prevalence
of HBV has been decreasing in developed countries.
The hepatitis B virus is primarily found in blood of infected individuals. Virus titres,
as high as ten billion virions per millilitre of blood, have been reported in HBe-positive
carriers. However, HBV has also been detected in other body fluids including urine,
saliva/nasopharyngeal fluids, semen, and menstrual fluids.2,3 This virus has not been detected
in faeces, perhaps due to inactivation and degradation within the intestinal mucosa or by the
bacterial flora.4
Acute hepatitis B is still a major problem in the United States with 150-450,000
persons newly infected each year. The US Centers for Disease Control and Prevention (CDC)
collects data on the incidence of acute hepatitis in 4 sentinel countries throughout the US.
Data from these countries have been collected for more than a decade and provides a useful
indication of changes in the incidence of all types of hepatitis and associated risk factors.
The National Health and Nutrition Surveys (NHANES II and III) also collected serum
for hepatitis markers. The prevalence of all markers of hepatitis B infection was 4.9% of the
population and 0.4% for evidence of chronic hepatitis B infection.
Risk factors for hepatitis B infection have evolved over the last 20 years with a
majority of new cases occurring from sexual transmission. High-risk heterosexual activity
accounted for 45% of new cases with an additional 15% in men who have sex with men.
Injection drug use now accounts for only 21% of new cases of hepatitis B.

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Hepatitis B infection is on the rise in India with 43 million people identified as carriers of the
disease, according to a gastroenterologist here. The virus, considered 100 per cent more
infectious than AIDS, spreads through blood and body fluid contents with every five in 100
people in India getting infected.
We report here a case of hepatitis B positive with symptoms responded to a treatment
with a polyherbal formulation HD-03/ES.

CASE HISTORY
Twenty-nine year young married female was suffering from repeated attacks of low-grade
fever, generalized weakness, headache, loss of appetite and loss of weight for more than two
years. She was treated randomly at various places for generalized weakness, which included
tonics, vitamins and minerals as oral supplements as well as injections. The history revealed
female had delivered a baby boy 4 years ago, since then she suffered post-partal weakness
and treated conventionally from time-to-time.
There were no signs of any physical or clinical improvement, made her undergo
complete physical and biochemical investigations for suspected hepatitis about 8 months
back. This revealed mild anaemia and along with positive evidence of hepatitis B surface
antigen (HBsAg). She was referred to us for the possible treatment with herbal drug. We
subjected her complete investigations.

INVESTIGATIONS
Considering the possibility of administering our new research drug HD-03/ES, we subjected
her for a complete haematological, biochemical and immunological investigations, which
included determination of HBsAg, polymerase chain reaction (PCR), along with liver
function test. We obtained her consent for undergoing the new drug treatment along with
repeated investigations necessary from time-to-time to evaluate the response.
We administered her HD-03/ES at a dose of 2 capsules (500 mg), twice daily. She is
continuing the treatment for past 5 months. We have been monitoring her every month with
respect to physical, biochemical and immunological investigations.

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RESULTS
She has been continuously taking HD-03/ES without any hesitation, indicates the positive
compliance to HD-03/ES. She reported feeling of well-being, improvement of appetite and
weight gain of about 6 kgs.
SURASE ELISA test revealed 48.60% reduction of HBsAg and the ELISA tiiter was
reduced from 1.442 to 0.741 OD values at the end of 4 months. The subsequent readings at
5th and 6th months are not available due to non-availability of ELISA reader. The
chronological details of the results are given in Table 1.

Table 1 HBsAg and PCR results treated with HD-03/ES

Sample Particulars ELISA Titer % Reduction PCR

(HBsAg) (HBsAg) Results
Before Treatment 1.442 - ++++
1 months 1.487 Nil +++
After Treatment 2 months 0.748 48.13 ++
3 months 0.745 48.62 -
4 months 0.741 48.60 -

Note: ‘+’ Indicates the intensity of PCR amplified HBV specific 523 bp DNA fragment

The PCR analysis using the amplification of 523 bp HBV specific DNA fragments revealed
disappearance of viral DNA at 3rd month onwards (Figure 1). HD-03/ES did not produce
adverse effects on the liver functions (Table 2).

Table 2 Liver function tests

Parameters Before After treatment
Treatment
1 2 3 4 5 6
month Months Months Months Months Months
 Total Protein (gm/dl) 6.46 6.60 6.40 6.80 7.10 7.30 7.10
 Albumin (gm/dl) 3.97 4.10 3.90 3.90 3.90 3.70 4.30
 Globulin (IU/L) 2.46 2.50 2.50 2.90 3.20 3.60 2.80
 SGOT (IU/L) 10.00 15.00 18.00 21.00 21.00 20.00 9.00
 SGPT (mg/dl) 14.00 11.00 10.00 17.00 7.00 9.00 20.00
 Total bilirubin (mg/dl) 0.71 0.61 0.67 0.56 0.53 0.53 0.51
 Direct bilirubin (mg/dl) 0.17 0.16 0.20 0.10 0.10 0.15 0.11
 Indirect bilirubin (mg/dl) 0.54 0.45 0.47 0.46 0.43 0.38 0.40

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The results indicated the significant reduction of HBsAg along with disappearance of viral
DNA suggesting positive effect of HD-03/ES.

Figure 1: PCR results before and after treatment with HD-03/ES

DISCUSSION

HD-03/ES is a research polyherbal formulation containing principle herbs such as Cyperus

rotundus and Cyperus scariosus (125 mg each). The roots and rhizomes of Cyperus rotundus
is used in different diseases like chronic diarrhoea, inflammation, skin rashes, and excess
bleeding. It has also antiestrogenic, antimicrobial, anthelmintic, antihistaminic, antiemetic,
antipyretic, and antidiabetic activities5. The roots of Cyperus scariosus have a folkloric
reputation as a cordial, tonic desicant, emmenagogue, diaphoretic and diuretic. It remained to
be an important ingredient of several prescriptions used in indigenous system of medicine to
treat a variety of diseases including diarrhoea, epilepsy, fever, gonorrhoea, syphilis, and liver
damage6.
In vitro studies have been conducted in the PLC/PRF5 cells and in the hepatitis B
virus transfected HepG2.2.2.15 cell line for assessing the suppression of HBsAg by HD-
03/ES. After 48 hours of incubation, the cells were treated with the filtered extract of HD-
03/ES at a concentration of 200 µg/ml and incubated atmosphere under 5% CO2. The

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suppression of HBsAg was estimated by a sensitive ELISA system and using an ELISA
reader (Anthos II). HD-03/ES produced 94% suppression of HBsAg.
The HepG2.2.2.15 cells were treated with the HD-03/ES for 12 days to study the
effect on long-term incubation of the plant extract on the viral DNA. The media was replaced
with fresh DMEM containing 200 µg/ml of the plant extract once in three days. At the end of
the Day 12, the cells were taken for DNA isolation by guanidine thiocyanate followed by
Phenol Chloroform extraction method. The DNA isolated was then used for PCR
amplification using of HBV specific 523 primer of viral DNA fragment. The amplified PCR
products were run on a 2% agarose gel and the DNA pattern were visualized on a gel
documentation system. The PCR study indicated the elimination of viral DNA in the cells
treated with HD-03/ES compared to the control group.
The in vitro efficacy of HD-03/ES in terms of reducing viral load and inhibition viral
load and inhibition of HBsAg confirms our finding in this patient treated with HD-03/ES at
the dose of 2 capsules (500mg) twice daily. This patient being a chronic carrier of hepatitis B
may require prolonged treatment for complete suppression of HBsAg. Since this patient has
not received any conventional anti-viral drug or interferon, suggests the positive effects are
definitely produced by HD-03/ES.
We have initiated the controlled clinical trials using HD-03/ES in acute and
chronic carriers of hepatitis B. The interim results of these trials are encouraging.

ACKNOWLEDGEMENT
We appreciate and acknowledge Dr. Rangesh Paramesh, MD (Ayu.), Senior Medical
Advisor, R & D Center, The Himalaya Drug Company, for making this formulation available
for clinical use.

References
1. Mosley JW. The Epidemiology of viral Hepatitis: An Overview Am J Med Sci 1975;
270: 253.
2. Alter HJ, Purcell RH, Gerin JL. Et al. Transmission of Hepatitis B Surface Antigen-
Positive Saliva and Semen. Infect Immun 1977; 16: 928.

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3. Davison F, Alexander GJM, Trowbridge R, et al. Detection of Hepatitis B Virus DNA

in Spermatozoa, Urine, Saliva and Leucocytes of Chronic HBsAg Carriers. Hepatology
1987; 4: 37.
4. Grabow WOK, Prozesky OW, Applebaum PC, et al. Absence of Hepatitis B Antigens
from Feces and Sewage as a Result of Enzymatic Destruction. J Infect Dis 1975; 131:
658
5. Pal DK, Dutta S. Evaluation of the Antioxidant Activity of the roots and rhizomes of
Cyperus rotundus. Indian Journal of Pharmaceutical Sciences 2006; 257.
6. Anwar HG, Janbaz KH, Zaman M, Lateef A, Tariq SR, Ahmad HR. Hypotensive and
Spasmolytic Activities of Crude Extract of Cyperus scariosus. Arch Pharm Res 1994;
145-149.

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