The use of chemometric modelling to determine chemical composition-antimicrobial activity relationships of essential oils used in respiratory tract infections.

The antimicrobial effects of essential oils are commonly cited within aromatherapeutic texts for use in respiratory tract infections. These essential oils are inhaled or applied to the skin to treat infections and manage symptoms associated with these conditions. A limited number of these essential oils have been scientifically studied to support these claims, specifically, against respiratory pathogens. This study reports on the minimum inhibitory concentration (MIC) of 49 commercial essential oils recommended for respiratory tract infections, and identifies putative biomarkers responsible for the determined antimicrobial effect following a biochemometric workflow. Essential oils were investigated against nine pathogens. Three essential oils, Amyris balsamifera (amyris), Coriandrum sativum (coriander) and Santalum austrocaledonicum (sandalwood) were identified as having greater activity (MIC value = 0.03-0.13 mg/ml) compared to the other essential oils investigated. The essential oil composition of all 49 oils were determined using Gas Chromatography coupled to Mass Spectroscopy (GC-MS) analysis and the GC-MS data analysed together with the antimicrobial data using chemometric tools. Eugenol was identified as the main biomarker responsible for antimicrobial activity in the majority of the essential oils. The ability of a chemometric model to accurately predict the active and inactive biomarkers of the investigated essential oils against pathogens of the respiratory tract was 80.33%.

Thesium chinense Turcz.: An ethnomedical, phytochemical and pharmacological review.

ETHNOPHARMACOLOGICAL RELEVANCE: Thesium chinense Turcz. has been used to treat mastitis, pulmonitis, tonsillitis, iaryngopharyngitis and upper respiratory tract infections in the indigenous medicine of China for a long history. Presently, several pharmaceutics prepared by this medical herb have been clinically used for the therapy of infectious diseases. AIM OF THE REVIEW: This review aims to comprehensively summarize the current researches on the ethnomedical, phytochemical and pharmacological aspects of T. chinense, and discuss their possible opportunities for the future research. MATERIALS AND METHODS: Extensive database searches, including Web of Science, SciFinder, Google Scholar and China Knowledge Resource Integrated, were performed using keywords such as 'Thesium chinense', 'Bai Rui Cao', and their chemical constituents. In addition, local classic herbal literature on ethnopharmacology and relevant textbooks were consulted to provide a comprehensive survey of this ethnomedicine. RESULTS: Thirty four chemical constituents, including flavonoids, alkaloids, and terpenoids, have been identified from T. chinense. Of which, flavonoids are the predominant and characteristic constituents. The crude extracts, the purified constituents, and commercial available pharmaceutics have displayed diverse in vitro and in vivo pharmacological functions (e.g. anti-inflammation, antimicrobial activity, analgesic effect, hepaprotection), and are particularly useful as a potential therapeutic agent against inflammation-related diseases. CONCLUSIONS: T. chinense is an important ethnomedical medicine and possesses a satisfying effect for treating inflammation, microbial infection, and upper respiratory diseases. It has received plenty of researches on its phytochemical and pharmacological aspects since 1970s. These findings definitely establish the link between chemical composition and pharmacological application, and support the ethnomedical use of T. chinense in the indigenous medicine of China. However, chemical composition of this plant and the molecular mechanisms of purified constituents have not been comprehensively investigated, and thus the trace constituents and the therapeutic targets of bioactive constituents deserve a further exploration. Collectively, the researchers should pay more attention to a better understanding and application of this ethnomedical plant.

A review of the ethnobotany, contemporary uses, chemistry and pharmacology of the genus Thesium (Santalaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Many plant species representing the hemi-parasitic genus Thesium play important roles in communities around the globe as evidenced by the numerous ethnobotanical and contemporary uses, and pharmacological activities. However, no attempt has been made to amalgamate and analyze all of the available information. A comprehensive survey is needed to highlight knowledge gaps, as well as to determine the economic importance and commercial potential of the genus. AIMS OF THIS REVIEW: To provide a comprehensive report on the species diversity, geographical distribution, ethnobotany, contemporary uses, chemistry, pharmacology and toxicology of the genus Thesium, as well as to give insights into possible future research opportunities. MATERIALS AND METHODS: Literature on the ethnobotany, contemporary uses, chemistry and pharmacology of Thesium was gathered from standard search engines (Google, Google Scholar, PubMed, SciFinder and Scopus) using the phrase Thesium, as well as generic synonyms. Additional information came from relevant books, theses, patents and label information from herbarium specimens in the National Herbarium in Pretoria, South Africa. Information on geographical distributions was compiled from regional floras, regional revisions, original species descriptions and databases (GBIF, IPNI, Plants of the World online and The Plant List). Scientific names and synonyms were validated through www.plantsoftheworldonline.org. RESULTS: A total of 23 Thesium species, 17 from Africa and six from Asia, were found to have traditional and contemporary uses. Despite the near cosmopolitan distribution of the genus, no uses were recorded for Europe, Australia and North America. Thesium plants are most commonly used as medicines (18 species), functional foods and beverages (seven species), charms (six species) and crafts (three species), but also have several other minor uses. Charm uses were restricted to southern and East Africa, while several contemporary uses such as functional feeds and fodders, growth mediums and fertilizers and veterinary medicine were unique to Asia. Thesium chinense is by far the most utilized and versatile species with a total of 173 uses in nine use categories, followed by T. longifolium with 39 uses in six use categories. No specific trends were observed in the plant parts used. As a medicine Thesium is used to treat 137 ailments, predominantly reproductive and breast (22), respiratory tract (18), degenerative (11), digestive (11) and urinary (10) ailments. Chemical analyses are available for only eight species, with flavonoids, fatty acids and alkaloids as the main compounds. The potential influence of the host plants on the chemistry and pharmacology remains unexplored. The pharmacological activities of two species, T. chinense and T. viride, have been studied, while three other species are reported to be poisonous. Thesium chinense has analgesic, anti-inflammatory, anti-oxidation, chemopreventive, cytotoxic and other general therapeutic properties, and T. viride anti-bacterial activity. CONCLUSION: This study has highlighted the ethnobotanical, contemporary and pharmacological importance of Thesium and informed possible future research opportunities. While ample information is available on the traditional uses of the richly diverse African Thesium species, the few Asian species dominate the literature on contemporary uses and pharmacology, while relevant literature on species in the rest of the world is altogether lacking. In light of the popularity of Asian species as ingredients in contemporary medicines and products, further research is needed into African species and their uses, including potential commercial uses. As an important medicinal hemi-parasite in both Africa and Asia, the identities of ethnobotanically relevant species and their phytochemistry, pharmacology and toxicology remain underexplored and require more research attention.

Decolorization affects the structural characteristics and antioxidant activity of polysaccharides from Thesium chinense Turcz: Comparison of activated carbon and hydrogen peroxide decolorization.

Different decolorization methods have different effects on the properties of polysaccharides. In this study, polysaccharides extracted from Thesium chinense Turcz (T. chinense) were decolorized using either hydrogen peroxide (H2O2), which has been previously reported, or activated carbon. To begin, the conditions for decolorization by activated carbon were optimized through single factor experiments and orthogonal modeling and were predicted to be best at 0.5% (w/w) activated carbon, pH 5.0 and 40 °C in a water bath for 60 min. Following treatment by the two methods, the structures of the decolorized polysaccharides were compared. The samples treated by activated carbon (CTP) had higher molecular weight (Mw: 3.064 × 105 g/mol) and more sulfate groups (14.33%, w/w), whereas the samples treated by H2O2 (HTP) had lower Mw (8.349 × 104 g/mol) and more uronic acid (35.02%, w/w). The antioxidant capacities of the two samples were analyzed by free radical scavenging (DPPH, hydroxyl and ABTS) and cell antioxidant activity (CAA). The HTP showed a better scavenging effect on free radicals and a higher CAA. Together, the results showed that various decolorization methods differentially influence the characteristics of the polysaccharides extracted from T. chinense.

Evaluation of In Vivo Antidiarrheal Activity of 80% Methanolic Leaf Extract of Osyris quadripartita Decne (Santalaceae) in Swiss Albino Mice.

The leaf of Osyris quadripartita is traditionally used for the management of diarrhea in different parts of Ethiopia. However, its use has not been scientifically validated for its efficacy. The aim of this study was to investigate antidiarrheal activity of hydroalcoholic leaf extract of O. quadripartita in mice models. Different doses of the methanolic leaf extract of O. quadripartita (100, 200, and 400 mg/kg) were tested for antidiarrheal activity using castor oil-induced diarrhea, enteropooling, and gastrointestinal motility models in Swiss Albino mice. The activities of the extract at different doses were compared with standard drugs and negative control groups of mice. The extract at all tested doses resulted in significant reduction ( P < .01) in number of wet feces, whereas significant reduction ( P < .01) in frequency of defecation in castor oil-induced diarrhea was seen at a dose of 400 mg/kg. It also showed a dose-dependent and significant reduction of volume of intestinal content in the enteropooling model at all tested doses and the observed results in 200 and 400 mg/kg were better than the standard drug, loperamide. However, significant antimotility effect was not observed at any of the tested doses. From these results we can conclude that methanolic leaf extract of O. quadripartita showed antidiarrheal activity.

Effect of crude leaf extract of Osyris quadripartita on Plasmodium berghei in Swiss albino mice.

BACKGROUND: Continuous emergence of multi-drug-resistant malaria parasites and their rapid spread across the globe warrant urgent search for new anti-malarial chemotherapeutics. Traditional medicinal plants have been the main sources for screening active phytochemicals against malaria. Accordingly, this study was aimed at evaluating the anti-malarial activity of Osyris quadripartita Salzm. Ex Decne., a plant which is used for traditional malaria treatment by local people in different parts of Ethiopia. METHODS: Aqueous, chloroform and methanol crude leaf extracts of the plant have been prepared and tested for acute toxicity and anti-malarial efficacy in Plasmodium berghei (ANKA strain)-infected Swiss albino mice. RESULTS: At three oral doses of 200, 400 and 600 mg/kg the plant material was safe, chemosuppressive and thus prevented body weight loss, hematological abnormalities and increased mice mean survival time compared to the negative control. The most efficacious extract was that of chloroform which prolonged mean mouse survival past day 11 of infection with all the mice in this group having the highest parasitemia suppression rate (41.3%, at 600 mg/kg) although parasite clearance was not achieved compared to the standard drug (chloroquine) against the parasite. CONCLUSION: The finding supports the traditional use of the plant for the treatment of malaria. However, further confirmatory studies followed by isolation and characterization of the active anti-malarial compound (s) of the plant that is/are responsible for the observed parasite suppression is needed before it is recommended for malaria drug search and discovery.

[Study on winter dormancy of Thesium chinense and its phenological phase].

In order to explore reasonable artificial cultivation pattern of Thesium chinense, the biological characteristics and nutrients change in the process of winter dormancy of T. chinense was studied. The phenological period of T. chinense was observed by using fixed-point notation and the starch grains changes were determined dynamically by PAS-vanadium iron hematoxylin staixjing method. Soluble sugar and starch content were measured by anthrone-sulfuric acid method and amylase activity was determined by DN'S method. The results showed that the normal life cycle of T. chinense was two years. T. chinense was growing by seed in the first year, but growing by the root neck bud in the second year. During the process of dormancy, starch and soluble sugar could mutual transformation in different periods. T. chinense had sufficient carbohydrate to maintain growth and also a lot of small molecules to improve their ability to fight against adversity.

Unprecedented furan-2-carbonyl C-glycosides and phenolic diglycosides from Scleropyrum pentandrum.

Five unprecedented furan-2-carbonyl C-glycosides, scleropentasides A-E, and two phenolic diglycosides, 4-hydroxy-3-methoxybenzyl 4-O-ß-D-xylopyranosyl-(1→6)-ß-D-glucopyranoside and 2,6-dimethoxy-p-hydroquinone 1-O-ß-D-xylopyranosyl-(1→6)-ß-d-glucopyranoside, were isolated from leaves and twigs of Scleropyrum pentandrum together with potalioside B, luteolin 6-C-ß-D-glucopyranoside (isoorientin), apigenin 8-C-ß-D-glucopyranoside (vitexin), apigenin 6,8-di-C-ß-D-glucopyranoside (vicenin-2), apigenin 6-C-α-L-arabinopyranosyl-8-C-ß-D-glucopyranoside (isoschaftoside), apigenin 6-C-ß-D-glucopyranosyl-8-C-ß-D-xylopyranoside, adenosine and l-tryptophan. Structure elucidations of these compounds were based on analyses of chemical and spectroscopic data, including 1D and 2D NMR. In addition, the isolated compounds were evaluated for their radical scavenging activities using both DPPH and ORAC assays.

[Study on quality of medicinal material of Thesium chinense].

OBJECTIVE: To study the quality of medicinal material of Thesium chinense. METHOD: The active ingredients of total flavonoids, kaempferol, mannitol, polysaccharides were selected as the evaluative indicators to comprehensively evaluate the quality of medicinal material of T. chinense. RESULT: The total flavone 3.38%, kaempferol 0.984 7 mg x g(-1), mannitol 6.12%, polysaccharides 14.08% and water soluble extract 30.28% were measured in the sample of T. chinense. There was a certain correlation among these selected active ingredients. The compositions of most biological activities component in T. chinense of different populations were similar, but the contents were different significantly. The content of activities component in T. chinense showed a general tendency of declining during growth period and with the increase time of storage. CONCLUSION: Flavone, kaempferol, mannitol, polysaccharides should be selected as the indicators to evaluate the quality of medicinal material of T. chinense.

[Determination and principal components analyze of mineral elements in different population of Thesium chinense].

OBJECTIVE: To determine the concentration of mineral elements and analyze the principal components in Thesium chinense. METHOD: Mineral elements were determined by ICP-DES. Principal components analysis (PCA) was used toanalyze and evaluate the characteristic elements. RESULT: T. chinense contained more than 17 mineral elements, Fe, Zn, Cu, Mn, Cr, Co, Ni, Sr, B, Ca, P, K, Na, Mg, Mg, Mn, Zn and Cu were abundance in T. chinense. The concentration of Mn and Zn were about 100 microg x g(-1), Mg was from 1 898.0 to 3 137.73 micro x g(-1), Fe > 500 microg x g(-1). PCA concluded that four factors (F1, F2, F3, F4 ) could be used to evaluate the quality of T. chinense. The function is following: F = 0.444 77F1 + 0.237 71F2 + 0.167 28F3 + 0.150 24F4. The scores of different populations of T. chinens were from 0.627 to 0.189. Zn,Cu,Mg,P, K, Al, Fe, Cr, Na, Co, Pb, Mn, B and Ca were the characteristic elements of T. chinense. CONCLUSION: T. chinense contain more than 17 mineral elements. Zn, Cu, Mg, P, K, Al, Fe, Cr, Na, Co, Pb, Mn, B and Ca were the characteristic elements of T. chinense.

Exocarpic acid inhibits mycolic acid biosynthesis in Mycobacterium tuberculosis.

Exocarpic acid (13 E-octadecene-9,11-diynoic acid) from Exocarpos latifolius R.Br. (Santalaceae) was previously shown to have specific antimycobacterial activity. Microarray data suggested inhibition of fatty acid metabolism as a potential mode of action. Experiments designed to elucidate the mechanism of action showed that exocarpic acid was effective at inhibition of mycolic acid biosynthesis and did not act by dissipating the proton gradient in treated M. tuberculosis. Amide derivatives of exocarpic acid displayed similar properties to exocarpic acid, while other polyacetylenic fatty acids varied in their effects on mycolic acid biosynthesis.

[Study on the flavonoids compounds of Thesium chinese].

OBJECTIVE: To study the flavonoids compounds from the effective fraction of Thesium chinese in anti-inflammation. METHODS: The isolation and purification were carried out by many kinds of column chromatographic techniques. The flavonoids were analyzed by HPLC-DAD, and elucidated by 1H-NMR, 13C-NMR and ESI-MS. RESULTS: Five compounds were obtained and therir structures were determined as kaempferol-3-O-glucoside (I), rutinoside (II), luteolin-7-O-glucoside (III), kaempferyl 5-methyl ether (IV), apigenin-5-O-neohesperidoside (V). CONCLUSION: Compounds IV and V are isolated from this plant for the first time.

Antimycobacterial activity of Exocarpos latifolius is due to exocarpic acid.

Lipophilic fractions of stem extracts from Exocarpos latifolius, native to Papua New Guinea, showed significant activity against Mycobacterium tuberculosis H37Ra. Bioactivity-guided fractionation yielded exocarpic acid (E-octadeca-13-ene-9,11-diynoic-acid) as the major active component. Several new exocarpic acid analogs were also shown to be active. Exocarpic acid has previously been reported active against gram-positive, but not gram-negative bacteria. Work presented here demonstrates the selective activity of exocarpic acid against Mycobacterium tuberculosis H37Ra.

Radical scavenging activity and total phenolic content of extracts of the root bark of Osyris lanceolata.

The root bark of Osyris lanceolata was screened for its antioxidant potential using three variations of the DPPH radical scavenging method viz a TLC-autographic assay, a semi-quantitative TLC assay, and a spectrophotometric assay. The total phenolic content was evaluated as gallic acid equivalents (GAE) using the Folin-Ciocalteu method. The powdered root bark was extracted sequentially with n-hexane, chloroform, methanol and 90% methanol/water. A separate supercritical fluid extraction (SFE) of the root bark was also carried out. The radical scavenging ability, as measured by IC50 values, was found to be 48.4 +/- 0.4 and 49.5 +/- 1.9 microg/mL for the 90% methanol/water and methanol extracts respectively, while those of the chloroform, n-hexane, and SFE extracts were found to be > 250 microg/mL. Under the same conditions, the values for the standards, ascorbic acid and gallic acid, were found to be 38.70 +/- 0.08 and 2.86 +/- 0.01 microg/mL respectively. The radical scavenging power of the five extracts correlated positively with the total phenolic content and was ranked in the following decreasing order: 90% methanol/water > methanol > chloroform > SFE > n-hexane with total phenolic contents of 290.2 +/- 3.3, 271.0 +/- 3.7, 74.4 +/- 0.8, 62.5 +/- 0.4 and 49.5 +/- 1.9 mg GAE/g of dry extract, respectively. The 90% methanol/water and methanol extracts showed several components with high antioxidant activity displaying fast kinetics in both TLC assays, while the chloroform, SFE, and n-hexane extracts exhibited slow kinetics antioxidant activity.

Botanicals used in complementary and alternative medicine treatment of cancer: clinical science and future perspectives.

BACKGROUND: Botanicals and herbal combinations are among the most common complementary and alternative medicine (CAM) approaches used by cancer patients both for cancer treatment and management of cancer symptoms. Despite their widespread use, however, the safety and efficacy of many botanicals has not been established in controlled clinical trials. OBJECTIVES: This article reviews the published evidence for the safety and clinical benefit of botanicals used in the treatment of cancer and cancer symptom management and describes the continuing clinical trials of botanicals with applications in oncology. METHODS: Literature searches were conducted in PubMed, EMBASE, Cochrane Clinical Trials databases, Pharmaprojects and CRISP (Computer Retrieval of Information on Scientific Projects) clinical trials databases. CONCLUSION: A number of botanicals have shown promise for cancer symptom management but need further study. A limited number of multi-agent nutritional supplement approaches are being explored in clinical trials. Botanical immunomodulators and botanical products shown to affect pathways of angiogenesis, apoptosis and cell signaling in vitro have stimulated research interest and may broaden the range of available cancer treatments.

[Study on endogenesis inhibitory substances in seed of Thesium chinense].

OBJECTIVE: To investigate the activity and the location of the endogenesis inhibitory substances in seed of of Thesium chinense. METHOD: The rough extracts from different concentrations, places and extracting times of T. chinense seeds were prepared and determined by its effect on wheat and Brassica sp seed. RESULT AND CONCLUSION: The extract dramatically inhibited the germentation and the growth of wheat and Brassica sp seed and the inhibitory activity was positively related to the concentration of the extracts. When the concentration reached 0. 32 g mL(-1), wheat seed did not germentate. With increasing the extracting times, the inhibitory activity first decreased, then increased. The capsule and kernel both had inhibitory substances and the latter was stronger than the former. Highly active inhibitory substances were found first time in the seed of T. chinense.

Antiinflammatory and analgesic activities of Thesium chinense Turcz extracts and its major flavonoids, kaempferol and kaempferol-3-O-glucoside.

The ethyl acetate, chloroform extracts, and the two flavonoids kaempferol 1 and kaempferol-3-O-glucoside 2 isolated from whole plants of Thesium chinense Turcz were investigated for their antiinflammatory and analgesic activities. For the antiinflammatory activity, carrageenan-induced hind paw edema and xylene-induced mouse ear edema models, and for the analgesic activity, the acetic acid-induced abdominal constriction test was used. The ethyl acetate extract and two flavonoids showed significant (p<0.05 and p<0.01) and dose-dependent antiinflammatory and analgesic activity. The chloroform extract was inactive in the assay.

Comparative molecular and phytochemical study of the tree species Santalum austrocaledonicum (Santalaceae) distributed in the New-Caledonian archipelago.

We have tried to elucidate the origin of phytochemical variation in trees by studying concomitantly the chemical and microsatellite variations in Santalum austrocaledonicum. Eight natural populations were sampled in the New-Caledonian archipelago, a total of 157 individuals being analyzed. The main components, as revealed by gas chromatography (GC), were alpha- and beta-santalol (as in other sandalwood species), although the level of (Z)-lanceol was particularly high. Most of the chemical variation was observed within populations (83.7%). With microsatellites, the variation between populations was more pronounced (32% of the total variation). Although the chemical variation between populations was small, we investigated the effects of genetic drift and migration by comparing the chemical- and molecular-differentiation patterns. The poor congruence between neighbor-joining trees, confirmed by the non-significant Mantel test between the molecular and chemical distance matrices (R=0.26, P=0.12), showed that genetic drift and migration are not the main evolutionary forces acting on chemical differentiation between populations. We could not find any effect of soil and rainfall conditions neither. Although the impact of drift and migration cannot be discounted in rationalizing between-population differentiation, the low variation among populations could result from a stabilizing selection caused by the same phytopathogen charge across the natural range.

Antibacterial activity of plant extracts from northwestern Argentina.

AIMS: To determine the antibacterial and cytotoxic activities of aqueous and ethanolic extracts of northwestern Argentinian plants used in folk medicine. To compare the mentioned activities with those of five commercial antibiotics. To identify the compounds responsible for the antibacterial activity. METHODS AND RESULTS: Plant extracts were prepared according to traditional uses in northwestern Argentina. Antibacterial activity was assayed by agar dilution in Petri dishes and broth dilution in 96-well plates. Lethal dose 50 (LD(50)) was determined by the Artemia salina assay. Phytochemical analysis was performed by sample adsorption on silica gel, thin-layer chromatography (TLC), bioautography and UV-visible spectra. The results showed that Tripodanthus acutifolius aqueous extracts have lower minimal inhibitory concentrations (MIC) (502 and 506 microg of extracted material (EM) per ml for infusion and decoction, respectively) than cefotaxim MIC (640 microg ml(-1)) against Acinetobacterfreundii (303). These data were lower than their LD(50). Tripodanthus acutifolius tincture showed lower MIC (110 microg of EM per ml) and minimal bactericidal concentration (MBC) (220 microg of EM per ml) than cefotaxim (MIC and MBC of 320 microg ml(-1)) for Pseudomonasaeruginosa. This extract also showed a MIC/MBC of 110/220 microg of EM per ml, lower than oxacillin (MIC/MBC of 160/220 microg ml(-1)) for Staphylococcus aureus (ATCC 25923). The cytotoxicity of all extracts were compared with that of commercial antibiotics. Rutin (3,3',4',5,7-pentahydroxyflavone 3-beta-rhamnosilglucoside), iso-quercitrin (3,3',4',5,7-pentahydroxyflavone 3-beta-glucoside) and a terpene would be partially responsible for the antibacterial activity of T. acutifolius infusion. CONCLUSIONS: Tripodanthus acutifolius extracts had the ability to inhibit bacterial growth. The antibacterial activity differs with the applied extractive method, and it could be partially attributed to glycoflavonoids. This paper contributes to the knowledge of antibacterial capacity of plants from northwestern Argentina. SIGNIFICANCE AND IMPACT OF THE STUDY: These antibacterial activities support further studies to discover new chemical structures that can contribute to alleviate or cure some illnesses.

[Determination of astragulin in Thesium chinese by RP-HPLC].

OBJECTIVE: To establish a determination method of astragulin in Thesium chinese. METHOD: RP-HPLC analytical method was established using a Polaris C18 column and acetonitrile-water (23:77) as the mobile phase, with a flow rate of 10 mL x min(-1), detected at 346 nm. The method of sample is refluxing exation by 50% alcohol for 2 times. RESULT: The content of astragulin was from 0. 120% to 0. 155%, in different groups of T. chinese collected from the same location. CONCLUSION: The method was validated to show convenient and reliable.