Fitoterapia 75 (2004) 1423

Free radical scavenging activity of extracts prepared from fresh leaves of selected Chinese medicinal plants
Hu Fenglina,*, Lu Ruilia, Huang baoa, Ming Liangb
a

Anhui Agricultural University, Hefei 230036, PR China b Anhui Medical University, Hefei 230036, PR China Received 15 March 2003; accepted 28 July 2003

Abstract The free radical scavenging activity of the 80% methanolic extracts from fresh leaves of 300 Chinese medical woody plants was assessed with the aid of the stable DPPH radical. Among the plants screened, 56 species had strong free radical scavenging capacities, with IC50 values lower than 0.5 mg leaves per milliliter. Analysis of the medical uses of these plants showed that most of them are employed for their effects on hemostasis, as antiinflammatory, antimicrobial or for treatment of dysentery. These uses may be directly linked to the content in tannins and flavonoids and consequently to their free radical scavenging activities. 2003 Elsevier B.V. All rights reserved.
Keywords: Chinese medicinal plants; Free radical scavenging activity

1. Introduction The DPPH free radical scavenging assay is a simple and widely used screen for bioactive compound discovery. In conjunction with a microtitre plate reader, the assay can be easily carried out as a high througput test w1x. In the context of a screening of Chinese herbs for their free radical scavenging activities, we intend to assay most of the Chinese herbal plants. More than 8000 plant species, including
*Corresponding author. Fax: q86-551-2843286. E-mail address: hufenglin@hotmail.com (H. Fenglin). 0367-326X/04/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2003.07.003

H. Fenglin et al. / Fitoterapia 75 (2004) 1423

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more than 2000 woody plants are used or have been used at one point in time as medicinal plants. However, most of them have not been studied for their free radical scavenging activities w2x, even though taxonomically related species from Europe, America and Japan may have been studied intensively w3x. The phytochemistry and bioactivities of the majority of the locally used medical herbs of China have not been studied. The same is true for the non-used plant parts of many famous Chinese medicinal plants. For example, the root bark of Paeonia suffructicosa is well studied in the traditional Chinese Medicine w4,5x, but there are no reports about the free radical scavenging activity and chemical composition of the fresh leaves. We here report our findings from a screening of leaf extracts from more than 300 woody plants used as local or generally recognized medicinal herbs. 2. Experimental 2.1. General 1,1-Diphenyl-2-picryhydrazyl (DPPH) was bought from Aldrich (Shanghai). Analytical grade methanol was from Anhui Reagents Company (Hefei, China). A Multiscan (Ascent) microtiter plate reader was purchased from Thermo Bioanalysis Company (Helsinki, Finland). 2.2. Plants Fresh leaves from more than 300 species woody medicinal plants were collected in the botanical garden of Anhui Agricultural University and in the montain area of Anhui Province in June 2002. The fresh leaves were crushed and extracted for 48 h at room temperature with 80% MeOH. For every milligram of fresh leaves, 10 ml were added. Extraction was carried out under shaking with 80 rev. y min. The suspensions were centrifuged, and the supernatant diluted at different concentrations, which were used as test samples. 2.3. Quantitave analysis Quantitative measurement of radical scavenging properties was carried out in a 96 wells microtitre plate assay w1x. The reaction mixture contained 100 ml of test samples (or 80% MeOH as a blank) and 100 ml of a 1 mM solution of DPPH in EtOH. 100 ml of 10 mM solutions of ascorbic acid was used as positive control. Decoloration was measured at 517 nm after incubation for 20 min. Measurements were performed at least in triplicate. The actual decrease in absorption induced by the test compounds was compared to that of the positive controls. IC50 values calculated denote the concentration (milligram leaves per millilitre of 80% MeOH) of sample required to scavenge 50% of DPPH radical. 3. Results and discussion More than 300 medicinal plants were screened for DPPH radical scavenging activity according to the method described above. Among these, 56 species afforded

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Table 1 Free radical scavenging capacity of extracts, medicinal uses of herbs, and known constituents in leaves Name Plant parts used in Chinese medicine and their effects andyor uses Seed: hernia, ejaculatio nocturna, vitiligo, and acute bark, leaves: contact dermatitis, cuts, beriberi, burns Root bark: tinea, scabies and traumatic injuries; Leaves: hemoptysis, hematemesis Known constituents of the leaves Hinokiflavone sotetsuflavone kayaflavone6 Ponasterone A, makisterones, podocarpusflavones w2,6,7x Myricitrin, cannabiscitrin, myricetin w6,7x Juglone, ellagic acid, ascorbic acid w6,7x Casuarinin, chestannin, grandinin w6,7x Tannin w6x Tannin w6x Tannin w6x IC50 mgyml 0.483"0.036

Cunninghamia Lanceolata (Lamb.) Hook.f. Podocarpus macrophyllus (Thunb.) D.Don Myrica rubra (Lour.) Sieb. et Zucc. Platycarya strobilacea Siebet Zucc. Castanea mollissima Bl. Castanea seguinii Dode Lithocarpus glaber (Thunb.) Nakai Quercus acutissima Carr. Lindera reflexa Hemsl. Phoebe sheareri (Hemsl.) Gamble Nandina domestica

H. Fenglin et al. / Fitoterapia 75 (2004) 1423

0.421"0.014

Bark, root: dysentery, stomachache, vomiting, hernia, bleedings, traumatic injuries, toothache, burns, scalds, malignant furuncle, leprosy Leaves: furuncle; Fruit: distending pain in chest and hypochondrium, abdominal pain, arthralgia, carbuncle, tinea and scabies. Fruit shell: intestinal hemorrahage. Bark: erysipelas. Leaves: pertussis Root: insomnia, pneumonia, tuberculosis; Bark: erysipelas and furuncle. Inner layer of bark: ascites Fruit, Fruit shell: dysentery, hemorrhoidal bleeding and prolapse of the anus; Bark: for dysentery, scrofula, furuncle. Root bark: stomachache, tinea, scabies, allergic dermatitis, incised wound. Wood, branches and leaves: cholera, abdominal distension, edema.; Root: traumatic injuries. Root, stem and fruit: relieving cough and asthma,

0.330"0.009

0.487"0.037 0.319"0.018

0.345"0.022 0.490"0.028 0.358"0.011

Unknown Unknown

0.412"0.026 0.476"0.036

Domesticine,

0.404"0.013

Table 1 (Continued) Name Plant parts used in Chinese medicine and their effects andyor uses bactericidal; Laves: pertussis, cold with headache, inflammation and cough Root bark: analgesic, sedative, antipyretic, promoting blood circulation Seeds: cure pharyngitis, laryngitis, stomachache Flowers: haemostatic Whole herb: infections of the upper respiratory tract, hepatitis, dysentery and nephritis; Root: to invigorate blood circulation, promote lactation, diuresis. Root: cure exhaustion of vital essence and energy of the body due to overstrain Leaves: cure deficiency of vital energy; Root: cure exhaustion of vital essence and energy of the body due to physically overwork Fuit: a famous drug for removing obstruction in channels and promoting blood circulation; Resin: famous drug for traumatic hemorrhage; Root: rheumatic arthritis, toothache; Leaves: enteritis, dysentery, stomachache Root, leaves, flowers: hemostatic, antiinflammatory and bactericidal. Root: cure edema of hands and feet. Known constituents of the leaves glycosyloxyman. delonitrile, mag noflorine w2x Paeonoside Theanine w6x Caffeine, epicatechol w6x Unknown IC50 mgyml

Thunb. Paeonia suffruticosa Andr. Camellia oleifera Abel. Camellia japonica L. Hypericum patulum Thunb. Hamamelis mollis Oliv. Fortunearia sinensis Rehd.et Wils. Liquidambar formosana Hance

0.319"0.005 0.345"0.010 0.399"0.019 0.473"0.042

H. Fenglin et al. / Fitoterapia 75 (2004) 1423

Unknown Fortunearoside, bergenin w2x a- and b-pinene, camphene w2x

0.292"0.007 0.480"0.003

0.327"0.012

Loropetalum chinense R.Br. Distylium racemosum Sieb.et. Zucc Chaenomeles sinensis (Thouin) Koehne Chaenomeles

Quercetin, Unknown Unknown

0.345"0.017 0.300"0.004 0.456"0.043

Fruit: dysentery, arthralgia, spasm of muscle, swelling pain due to beriberi, vomiting, diarrhea. 17 Fruit: rheumatic arthritis, spasms, swelling pain due Unknown 0.383"0.039

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Table 1 (Continued) Name Plant parts used in Chinese medicine and their effects andyor uses to beriberi, vomiting, diarrhea. The fruit wine is a famous drug for arthralgia. Leaves: dysentery Fruit: cure indigestion and abdominal distension Fruit: cure diabetes, dysentery, spermatorrhoea; Root: parasiticide, diabetes. Root: rheumatic arthritis, injuries, prolapse of the anus, uterine prolapse; Leaves: carbuncles, furuncles, burns. Root bark: promoting blood circulation and restoring flow and reducing swelling. Flowers, root and leaves: stagnation menstruation, distending pain in the chest and abdomen, amenorrhea and swelling pain of scrofula with pus prior to rupture. Flowers: eliminating sputum, relieving cough, reducing edema, improving digestion. Whole plant: promoting blood circulation and dissipate blood stasis. Bark: promoting blood circulation, restoring menstrual flow, subduing swelling and removing toxic substances. Used to treat arthralgia, amenorrhea, inflammation of the throat, stranguria, subcutaneous swelling, ringworm, scabies, traumatic injuries and snakebite. Root and leaves: dysentery, jaundice, stranguria with turbid urine, leukorrhagia, commen cold, sore throat, toothache, carbuncle, furuncle, rheumatic arthralgia, Known constituents of the leaves IC50 mgyml

speciosa (Sweet.) Nakai Malus hupehensis (Pamp.) Rehd. Malus melliana (Hand.-Mazz.) Rehd Rosa cymosa Tratt. Rosa banksiae Ait. Rosa chinensis Jacq.

Unknown

0.422"0.028 H. Fenglin et al. / Fitoterapia 75 (2004) 1423

Isosakuranetin, Prunasin. Unknown

0.329"0.021 0.424"0.012

Unknown Rosamultin w6x

0.411"0.015 0.457"0.036

Kerria japonica (L.) DC. Rubus.trianthus Focke Cercis chinensis Bge.

Helenien, pectolinaroside eloxanthin w2,6x Unknown

0.398"0.014

0.440"0.018

Flavonoids, Tannin w6,8x

0.448"0.030

Glochidion puberum (L.) Hutch. traumatic injuries.

Tannin w6x

0.393"0.007

Table 1 (Continued) Name Plant parts used in Chinese medicine and their effects andyor uses Root, twig and leaves: CNS stimulant, promoting blood circulation, relaxing muscles and tendons, strengthening the spleen and tonifying the kidney. Used to treat lumbago due to pathogenic winddampness, numbness of the limbs, impotence, facial palsy, and the sequelae of poliomyelitis. Bark and leaves: detoxicant, parasiticide and analgesic. Curing lumbago and bites by rabid dog or poisonous snake, dispelling ascarides. Bark and leaves: tinea, scabies, schistosomiasis, furuncle, eczema. Fruit: cure abdominal pain Bark: drug for diarrhea, dysentery, enteritis, cholangitis, carbuncle, furuncle, eczema, scalds Shoot and leaves: drug for diarrhea, dysentery, sore throat, cancer sore in mouth, cabuncles, furuncles Bark, fruit: removing toxins, anti-inflammatory, hemostatic, analgesic Root: strengthen muscles, removing blood stasis; Leaves: parasiticide, hemostasis Leaves and buds: hypertension, antipyretic Known constituents of the leaves Securinine,rutin, allosecurinine, securinol A,B,C, dihydrosecurin, tannin w2,6,7x Unknown IC50 mgyml 0.473"0.023

Securinega suffruticosa (Pall.) Rehd.

H. Fenglin et al. / Fitoterapia 75 (2004) 1423

Alchornea davidii Franch. Sapium sebiferum (L.) Roxb. Euodia fargesii Dode Picrasma quassioides (D.Don)Benn. Pistacia chinensis Bunge Choerospondias axillaries (Roxb.) Burtt.et Hill. Toxicodendron sylvestre O.Kuntze Acer ginnala Maxim. ssp. theiferum (Fang) Fang Acer buergerianum Miq. Acer sinopurpurasces Cheng Acer pubipalma-

0.336"0.016

Isoquercitin, tannin, sprin w2,6x Unknown Picraquassioside, picrasin w6x Quercetin, catechin w2,6x Tannin w6x

0.455"0.032 0.379"0.015 0.361"0.026

0.295"0.009 0.477"0.019

Rhoifolin w6,7x Tannin w6x

0.390"0.015 0.272"0.004

Root: arthralgia Root: arthralgia Root: arthralgia, fractures, injury

Tannin w6x Unknown Vitexin w6,7x

0.326"0.003 0.343"0.008 19 0.281"0.013

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Table 1 (Continued) Name Plant parts used in Chinese medicine and their effects andyor uses Known constituents of the leaves Tannin w6x Elaeocarpusin w7x Methylgallate w6,7x Unknown IC50 mgyml

tum Fang Acer henryi Pax Acer nikoense Maxim. Koelreuteria paniculata Maxm. K. bipinnata Franch. var. integrifoliola (Merr.) T. Chen Xanthoceras sorbifolia Bunge Euscaphis japonica Dippel Lagerstroemia subcostata Koehne Lagerstroemia indica L.

Root: arthralgia Root: arthralgia Bloosom: anti-inflammatory and detumescence of eyes Root, flowers: antipyretic, relieve cough, parasiticide

0.428"0.030 0.304"0.013 0.441"0.027 0.339"0.033 H. Fenglin et al. / Fitoterapia 75 (2004) 1423

Wood, branches, leaves: rheumatic arthritis Fruit: analgesic, detumescent; Flowers: analgesic; Root: antirheumatic, invigorating the spleen Root, flowers: detoxicant, eliminating blood stasis. Root: carbuncles, furuncles and toothache; Leaves: dysentery, eczema and leeding injuries; Flowers: hemostatic, tinea and scabies; Bark, leaves and flowers are also laxative Leaves: detoxicant, wound treatment; Rind: chronic diarrhea and dysentery, prolapse of the anus, abdominal pain, metrorrhagia; Root: parasites, chronic diarrhea, chronic dysentery, and leukorrhea Twig, leaves and flowers: traumatic bleeding, bone fracture, dysentery. Flowers: irregular menstruation, traumatic injuries, rheumatism; Leaves: subcutaneous swelling, furuncle, carbuncle, traumatic and urticaria; Root:

Hexahydroxyflavanone w2x Euscamins w2,9x Tannin w6x Decinine, lagerine, decamine, decodine w2,6,7x Pelargonin, propenylpiperidine, asiatic acid, cralaegolic acid, casuarinin w2,3,6,7x Unknown

0.493"0.017 0.480"0.032 0.350"0.011 0.366"0.016

Punica granatum L.

0.283"0.003

Dendrobenthamia japonica (DC.) Rhododendron simsii Planch.

0.468"0.012

Matteucinol, matteucinin,

0.485"0.018

Table 1 (Continued) Name Plant parts used in Chinese medicine and their effects andyor uses hematemesis, epistaxis, irregular menstruation, metrorrhagia, dysentery, rheumatic pain, traumatic injuries. Whole plant: tuberculosis, tracheitis, hepatitis, orchitis Fruit, root and leaves: antipyretic, anti-inflammatory, detoxicant, treating carbuncles and subsidence of a swelling. Known constituents of the leaves ursolic acid andromedotoxin w2,6,7x Bergenin, quercitrin, myricitrin w2,10x Unknown 0.464"0.017 H. Fenglin et al. / Fitoterapia 75 (2004) 1423 IC50 mgyml

Ardisia japonica (Thunb.)Bl. Forsythia suspensa Vahl

0.437"0.025

21

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H. Fenglin et al. / Fitoterapia 75 (2004) 1423

extracts with IC50 values lower than 0.5 mg leaves per milliliter of 80% MeOH. The results of the screening are listed in Table 1, together with a short description of the traditional uses, used plant parts and known chemical constituents of the leaves. Among the 56 plants displaying pronounced radical scavenging activity, a significant number reportedly contain tannins or other polyphenols such as flavonoids. Although the secondary metabolites of many of the species screened are only known in part or have not even been studied up to now, it is likely that tannins are, at least in part, responsible for the strong free radical scavenging activities w11x. When considering the traditional uses of the plants listed in Table 1, it appears that a significant number have been employed to treat bleedings, dysentery, wounds and various skin afflictions. These uses may also relate to the presence of tannins. It is well known that tannins have anti-inflammatory, antidiarrheic and wound healing properties. Phenols such as methyl gallate possess antimicrobial properties, and physiological redox processes in the microbial cells may be disrupted by the strong reducing activity of tannins w12x. Furthermore, some tannin has been shown to act via inhibition of specific enzymatic functions. Elaeocarpusin, for example, inhibits AIDS virus replication, and casuarinin inhibits FAD oxidase and topoisomerase w7x. The anti-inflammatory properties of tannins are mainly due to their free radical scavenging activities, as free radical scavenders can stop some of the processes of inflammatory response w13x. Flavonoids are also widely encountered in the plants tested as the most active radical scavengers. Again, this is not surprising given that most flavonoids have strong free radical scavenging properties w13,14x. The multiple pharmacological properties of flavonoids, such as anti-inflammatory, antibiotic and cardiovascular activities w12x are, to a large extent, linked to their polyphenolic and hence radical scavenging nature w13x. Besides that, certain flavonoids such as hikinoflavone possess specific properties as antiviral or antitumoral compounds w7x. There is no doubt that tannins and flavonoids play important roles in the scavenging activities and medical uses of the plants studied here. The fact that these herbs certainly contain additional classes of secondary metabolites warrants further investigation. There is no information available on the chemical composition of leaf constituents for nearly one fifth of the plants listed. For another quarter of species, preliminary phytochemical studies have been carried out so far. Acknowledgments Thanks are due to Prof. Dr M. Hamburger, Institute of Pharmacy, FriedrichSchiller-University Jena, Germany, for suggestions and comments. References
w1x Cotelle JA, Bernier JL, Catteau JB, Pommery J, Wallet JC, Gaydou EM. Free Radical Biol Med 1996;20:35. w2x Huang TK, Ding ZZ, Zhao SX. Modern compendium materia medica. Beijing: Chinese Medical Science and Technology Press, 2001. p. 1.

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w3x Bruneton J. Pharmacognosy, phytochemistry and medicinal plants. Paris: Lavoisier Publishing, 1995. p. 1. w4x Lin HC, Ding HY, Wu YC. J Natl Prod 1998;61:343. w5x Liu F, Ng TB. Life Sci 2000;66:725. w6x Mi TY, Li CQ, Zhou HR, Qian XH. Anhui economical plants flora Anhui. Anhui Science and Technology Press, 1999. w7x Chen HF. Dictionary of plants bioactive compounds. Beijing: Chinese Medical Science and Technology Press, 2001. w8x Salatino A, Salatino ML, Giannasin DE. Biochem Syst Ecol 2000;28:545. w9x Takeda Y, Okada Y, Masuda T, Hirata E, Shinzato T, Takushi A, Yu Q, Otsuka H. Chem Pharm Bull 2000;48:752. w10x Piacente S, Pizza C, De Tommasi N, Mahmood N. J Natl Prod 1996;59:565. w11x Yokozawa T, Chen CP, Dong E. Biochem Pharmacol 1998;56:213. w12x Yao XS. Natural products chemistry. Beijing: Chinese Health Press, 1997. p. 241. w13x Sun CP, Zhang JZ, Duan SJ. Free radical biology. Anhui: Chinese Science and Technology University Press, 1999. w14x Hirano R, Sasamoto W, Matsumoto A. J Nutr Sci Vitaminol 2001;47:357.