Folk Medicinal Plant Research Articles

Development and validation of liquid chromatography-tandem mass spectrometry method for quantification of a potential anticancer triterpene saponin from seeds of Nigella glandulifera in rat plasma: application to a pharmacokinetic study.

Nigella glandulifera Freyn et Sit is a folk medicinal plant, whose seeds show significant anticancer activities attributed to triterpene saponins and volatile oil. In this study, an in vitro cytotoxicity assay demonstrated that Nigella A, the major component of triterpene saponins extracted from N. glandulifera, exhibited growth inhibition in the human lung carcinoma A-549 cell line. Due to this potential activity, a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify Nigella A in rat plasma for a pharmacokinetic study was developed. Nigella A and pravastatin, as internal standard (IS), were extracted from rat plasma using acetonitrile to precipitate protein. Separation was performed on an Agilent Zorbax SB-Aq (3.0 × 150 mm, 3.5 μm) column using a gradient elution method with acetonitrile-0.1% formic acid in water at a flow rate of 0.35 mL/min. Detection was performed using an electrospray ionization in a negative ion multiple reaction monitoring mode. The deprotonated precursor to product ion transitions monitored for Nigella A and IS was at m/z 1352.7→882.6 and m/z 423.1→321.0, respectively. The linear range was 0.240-120 μg/mL with a square regression coefficient (r=0.9996). The intra-day and inter-day precision was less than 6.93%. The simple extraction procedure provided recovery ranged from 92.32 to 95.44% for both analyte and IS. The method was proved to be reliable, precise, and accurate, and was successfully applied to a pharmacokinetic study of Nigella A in rats after i.v. administration via the tail vein at doses of 10, 20, and 30 mg/kg.

Flavonoid detection in hydroethanolic extract of Pouteria torta (Sapotaceae) leaves by HPLC-DAD and the determination of its mutagenic activity.

It is well known that phytotherapy has grown in popularity in recent years. Because a drug cannot be administered without ensuring its effectiveness and safety, the standardization and regulation of phytotherapeutic drugs are required by the global market and governmental authorities. This article describes a simple and reliable high-performance liquid chromatography-diode array detection analysis method for the simultaneous detection of myricetin-3-O-β-D-galactopyranoside, myricetin-3-O-α-L-arabinopyranoside, and myricetin-3-O-α-L-rhaminopyranoside present in the hydroethanolic extract (ethanol/H2O, 7:3, v/v) of Pouteria torta. The mutagenic activity of the extract was evaluated on Salmonella typhimurium and by an in vivo micronucleus test on the peripheral blood cells of Swiss mice. The linearity, sensitivity, selectivity, repeatability, accuracy, and precision of the assay were evaluated. The analytical curves were linear and exhibited good repeatability (with a deviation of less than 5%) and demonstrated good recovery (within the 83-107% range). The results demonstrate that the hydroethanolic extract exhibited a mutagenic activity in both assays, suggesting caution in the use of this plant in folk medicine.

Qualitative determination of bioactive metabolites through Q-TOF LC/MS in different parts and undifferentiated cultures of Ulmus wallichiana Planchon

Ulmus wallichiana is an endangered and folk medicinal plant used for healing fractures in India. Recently, the secondary metabolites isolated and identified from its bark revealed osteogenic activity. Considering the medicinal properties of its bark and the source for its identified and isolated metabolites, the requirement for production of alternative pathways was crucial. This study aimed to determine its bioactive metabolite using Quadrupole time-of-flight liquid chromatography-mass spectroscopy (Q-TOF LC/MS) in different parts of this plant with seasonal and altitudinal variability pertaining with the development of a protocol for induction of callus cultures and biomass production. Plant samples were collected during December–March, April–July and August–November in the year 2010–2012 from three different altitudinal locations (Nainital, Almora and Bageshwar districts) of Kumaon region, Uttarakhand, India. The results obtained from Q-TOF LC/MS analysis demonstrated the presence of four bioactive metabolites known for osteogenic activity in bark and stem samples round the year. Syntheses of bioactive metabolites in leaf samples were significantly affected by sampling season as well as altitudinal locations respectively. Callus derived from leaf explants was maintained in Murashige and Skoog medium containing (0.25 mg l−1) 2,4-Dichlorophenoxy-acetic acid (2,4-D) + (0.05 mg l−1) Kinetin and (0.25 mg l−1) α-Naphthaleneacetic acid + (0.5 mg l−1) 6-Benzylaminopurine in liquid phase showed optimal conditions for accumulating biomass and synthesis of identified bioactive metabolites.

Phenolic characterization and antimicrobial activity of folk medicinal plant extracts for their applications in olive production

Phytophthora spp is important in plant pathology due to the importance of the diseases it causes. In olive trees, severe damages are caused by the disease known as "dry branch" occasioned by Phytophthora nicotianae, P. citrophthora and P. palmivora. Much effort has been made to find efficient methods of control, with a low negative impact on environment. In this regard, treatment with plant extracts is a valid strategy. The aims of the present study are (i) to determine the polyphenol composition of extracts of Thymus vulgaris, Origanum vulgare, Matricaria recutita, and Larrea divaricata by CZE, (ii) correlate the analytical composition of these extracts with the inhibition on the mycelial growth, and (iii) determine the individual antimicrobial activity of the most active ingredients. A simple methodology was developed for the determination of catechin, naringenin, cinnamic acid, syringic acid, chlorogenic acid, apigenin, vanillic acid, luteolin, quercetin, and caffeic acid in plant extracts by CZE. The extraction of phenolic compounds in extract was performed by a miniaturized solid phase extraction using a home-made minicolumn packed with suitable filtering material (C18 , 50 mg). The optimized analyses conditions were: 30 mM boric acid buffer, pH 9.50; capillary, 57 cm full length, 50 cm effective length, 75 μm id, hydrodynamic injection 30 mbar, 2 s; 25 kV; 25°C, detection by UV absorbance at 290 nm. Sample results suggest that phenolic composition seems to have a great influence on inhibition of pathogens. The highest inhibitions of mycelial growth were observed for cinnamic acid and naringenin.

Study of Antisickling and Vasorelaxant Activities and Acute Toxicity Assessment of Crude Extracts of Leaves of Ficus sycomorus L. (Moraceae)

The leaves of Ficus sycomorus are used in Burkina Faso folk medicine for the treatment of sickle cell disease. The present comparative study of crude extracts of leaves (decoction, macerated extract and a 95% ethanol extract) was performed with the aim to assess the efficiency of this traditional use and to determine the most active of the three extracts. Antisickling activity was assessed by the Emmel's test. Vasorelaxant effect on rat aortic rings precontracted by phenylephrine with and without N-nitro-L-arginine methyl ester chloride (L-NAME) was also evaluated. The 95% ethanol extract (20 mg mL(-1)) showed the most antisickling activity on sickle erythrocytes, by inhibiting completely sickling of double heterozygote SC cells in 60 min and that of homozygote SS cells in 90 min. On the aorta this extract exhibited a significant (p<0.05) vasorelaxant activity, better than that of the other extracts, with an IC50 value of 6.86±0.13 mg mL(-1) against 18.78±0.38 and 28.56±1.27 mg mL(-1), respectively for the macerated extract and the decoction. When the aortic rings were pretreated with L-NAME, only the ethanolic extract conserved its vasorelaxant activity, up to 73% of relaxation. The acute toxicity of the decoction, assessed by intraperitoneal route and using the Litchfield and Wilcoxon method, led to an LD50 value of 1553.61 mg kg(-1) b.wt. This places the drug among those with low toxicity according to the WHO scale. These results confirm those previously obtained and provide a scientific basis supporting the use of this plant in folk medicine against sickle cell disease. They indicate the importance of Ficus sycomorus in the research of new antisickling molecules.

First Report on Natural Infection of Paeonia tenuifolia by 'Candidatus Phytoplasma solani' in Serbia.

Peony (Paeonia tenuifolia L.) is a herbaceous perennial plant known for its beautiful and showy flowers. In Serbia it is native to the Deliblato Sands and is used as an ornamental and medicinal plant in folk medicine. This plant species has become a rarity and for that reason peony was introduced into a botanical collection near Backi Petrovac (northern Serbia), where it has been maintained since 1988. Reddening of lower leaves observed on 10% of plants (5 of 50) in the collection at flowering in May 2012 gradually progressed throughout affected plants by the seed maturation stage. Five leaves from each of three reddened and three symptomless plants were sampled at the end of July 2012. Total nucleic acid was extracted separately from individual leaves (30 samples) using the CTAB (cetyltrimethylammonium bromide) method (2). A nested PCR assay using universal primer pairs P1/P7, followed by R16F2n/R16R2 (4), amplified 16S rDNA fragments of 1.8 and 1.2 kb, respectively. DNA from all three reddened plants (15 samples) yielded 1.2-kb amplicons after nested PCRs. Restriction fragment length polymorphism (RFLP) patterns obtained by digestion of nested products with endonucleases AluI, TruI, HpaII, or HhaI (Thermo Scientific, Lithuania) (4) were identical to those of the STOL reference strain included for comparative purposes, indicating that symptoms were consistently associated with plant infection by 'Ca. Phytoplasma solani' (Stolbur) phytoplasma. The 16S rDNA amplicons from two peony plants (1.2 kb from B15 and 1.8 from B18) were sequenced (GenBank Accession No. KC960487 and KF614623, respectively). BLAST analysis revealed a 100% identity between the sequences and GenBank sequences of Stolbur phytoplasma, subgroup 16SrXII-A phytoplasma, previously detected in maize (JQ730750) in Serbia and red clover (EU814644.1) in the Czech Republic. Phytoplasma associated diseases of other species of the genus Paeonia (P. lactiflora Pall. and P. suffruticosa Andrews) have been described elsewhere. Disease symptoms on P. lactiflora from Chile were associated with the phytoplasma that belongs to the ribosomal subgroup 16SrVII-A ('Ca. Phytoplasma fraxini') (1). Also, Stolbur phytoplasma from the 16SrXII group was detected on P. suffruticosa plants in China, manifesting yellowing symptoms (3). To our knowledge, this is the first report of naturally occurring Stolbur phytoplasma disease of P. tenuifolia L. in Serbia.

Simultaneous Determination of Capilliposide B and Capilliposide C in Rat Plasma by LC–MS/MS and its Application to A PK Study

Lysimachia capillipes Hemsl (Primulaceae), a folk medicinal plant in China, showed significant anti-tumor activity in recent studies. A reliable LC-MS/MS method was developed and validated for the simultaneous determination of capilliposide B and capilliposide C, the major bioactive components in this plant, in rat plasma. Rat plasma and whole blood samples were pretreated with dichlorvos, an esterase inhibitor, minimizing degradation of analytes in biological samples. The method validation was conducted over the curve range of 10.0 to 5000 ng/ml for both analytes. The intra- and inter-day precision and accuracy of the QC samples showed ≤6.1% RSD and 1.3-3.7% relative error. The method was successfully applied to determine the concentrations of capilliposide B and capilliposide C in incurred rat plasma samples, after administration of Lysimachia capillipes Hemsl extract for a rat PK study.

In vivo antimalarial efficacy of acetogenins, alkaloids and flavonoids enriched fractions from Annona crassiflora Mart.

Annona crassiflora and Annonaceae plants are known to be used to treat malaria by traditional healers. In this work, the antimalarial efficacy of different fractions of A. crassiflora, particularly acetogenin, alkaloids and flavonoid-rich fractions, was determined in vivo using Plasmodium berghei-infected mice model and toxicity was accessed by brine shrimp assay. The A. crassiflora fractions were administered at doses of 12.5 mg/kg/day in a 4-day test protocol. The results showed that some fractions from woods were rich in acetogenins, alkaloids and terpenes, and other fractions from leaves were rich in alkaloids and flavonoids. The parasitaemia was significantly (p < 0.05, p < 0.001) reduced (57–75%) with flavonoid and alkaloid-rich leaf fractions, which also increased mean survival time of mice after treatment. Our results confirm the usage of this plant in folk medicine as an antimalarial remedy.

A brief history of taxol

Traditional application of plant in folk medicine is a base for discovery of new active chemical substances. In the second half of 20th century, significant antitumor activity of extract from bark of the Yew tree was recognized. The compound that was responsible for this kind of properties has called taxol and had structure of diterpene with acyl groups. Low availability forced researchers to find different ways of taxol gaining. In the further years, many synthetic and semi-synthetic methods were developed. Nowadays, biotechnological methods with use of cell suspension cultures are introduced.

In vivo and in vitro anti-inflammatory properties of Achyrocline alata (Kunth) DC

Ethnopharmacological relevanceAchyrocline alata is a locally marketed (Mato Grosso do Sul/ Brazil) herb used in folk medicine as an anti-inflammatory and a sedative. Evaluate the anti-inflammatory properties of Achyrocline alata in both in vivo and in vitro models. Materials and methodsA hydroethanolic extract from inflorescences of Achyrocline alata (HEAa) was characterized by HPLC-DAD and compared to standards (chlorogenic acid; isoquercetrin; quercetin; 4,2׳,4׳-trihydroxy-6׳-methoxychalcone; gnaphalin; 3-O-methyl-quercetin; 3,5-dicaffeoyl-quinic acid and 4,5-dicaffeoyl-quinic acid). The in vivo anti-inflammatory properties of the HEAa (4, 20 and 100mg/kg, per os) were evaluated using the following animal models: carrageenan-induced paw edema in rats, carrageenan-induced vascular permeability and peritonitis in mice and an acetic acid-induced writhing model to test antihyperalgesic activity in mice. In vitro assays were performed to study the effects of the HEAa (0.16, 0.8 and 4mg/ml) on the cell viability, cell spreading and production of NO and H2O2 in stimulated macrophages. ResultsThe A. alata extract inhibited the development of edema and vascular permeability, reduced polymorphonuclear cell recruitment in the acute peritonitis assay and decreased the amount of writhing induced by acetic acid. The HEAa did not increase NO/H2O2 production, while it did inhibit production when the macrophages were stimulated by LPS or PMA at all tested concentrations. In the presence of HEAa, macrophage spreading did not increase even after stimulation with LPS. Additionally, the HEAa was nontoxic to macrophages at all tested concentrations. ConclusionsThe HEAa displayed anti-inflammatory and antihyperalgesic effects, which supports the use of this plant in folk medicine. These effects might be due to the flavonoids and phenylpropanoids derivatives present in the HEAa.

Natural phosphodiesterase-4 (PDE4) inhibitors from Crotalaria ferruginea

Bioassay-guided fractionation of the ethanol extract of the Chinese folk medicine Crotalaria ferruginea led to the isolation of a new isoflavonoid, 4′-hydroxy-2′-methylalpinum-isoflavone (1), and eight known analogs (2–9). Their structures were elucidated by spectroscopic analysis. Compounds 1, 2, 5, and 8 showed inhibitory activities against phosphodiesterase-4 (PDE4), a therapeutic target of asthma, with IC50 values ranging from 2.57 to 8.94μM. The possible action mechanism and the structure–activity relationship (SAR) of the active isoflavonoids were explored by using molecular docking and molecular dynamics (MD) simulation methods. Our study herein may explain the anti-inflammatory function of this plant in Chinese folk medicine.

An ethnopharmacological review on the Turkish Apiaceae species

Apiaceae is one of the richest families in the flora of Turkey. And it is also one of the most important families in which there are many popular plants used in traditional therapy in Turkey. The aim of this study is a revision on the Turkish folk medicinal plants of the Apiaceae family according to our investigations and scientific literature records. Our studies are based on mainly local ethnobotanical investigations. The ethnopharmacological information was obtained through open and semi-structured interviews from the local people. The specimens of the folk medicinal plants were collected during the field works and then identified. In addition, the scientific literature records on the subject were revised. According to the list based on our investigations and the literature records, 70 species of Apiaceae are used in therapy in Turkey. The plants are mostly used for the digestive system diseases, hemorrhoids, diabetes and as aphrodisiac and sedative

Ethno-Gynecological Disorders of Folk Medicinal Plants Used by Santhals of Dinajpur District, Bangladesh

Ethno-Gynecological Disorders of Folk Medicinal Plants Used by Santhals of Dinajpur District, Bangladesh

Prospecting for Bioactive Constituents from Traditional Medicinal Plants through Ethnobotanical Approaches

Pharmacologically active constituents from traditional medicinal plants have received great attention as sources of novel agents, pharmaceutical intermediates, and chemical entities for synthetic or semisynthetic drugs due to their potent pharmacological activities, low toxicity, and economic viability. Numerous components have been isolated from traditional medicinal plants, including alkaloids, flavonoids, and terpenoids, and clinical and experimental studies suggested that these components have useful pharmacological properties such as antiinfectious, antioxidative, and antiinflammatory effects. In this review, modern ethnobotanical approaches to explore folk medicinal plants as candidates for drug discovery with the greatest possibility of success are discussed. Determining the bioactive mechanisms and tracing structure-activity relationships will promote the discovery of new drugs and pharmacological agents.

Antimicrobial and Selected In Vitro Enzyme Inhibitory Effects of Leaf Extracts, Flavonols and Indole Alkaloids Isolated from Croton menyharthii

Croton species are used in folk medicine in the management of infections, inflammation and oxidative stress-related diseases. In order to isolate, characterize and evaluate the bioactive constituents of Croton menyharthii Pax leaf extracts, repeated column fractionation of the ethyl acetate fraction from a 20% aqueous methanol crude extract afforded three flavonols identified by NMR (1D and 2D) spectroscopic methods as myricetrin-3-O-rhamnoside (myricetrin, 1), quercetin-3-O-rhamnoside (2) and quercetin (3) along with an indole alkaloid, (E)-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine, [trans-N-(p-coumaroyl) serotonin, 4]. All the compounds are reported from the leaf extract of this plant for the first time. The crude extracts, four solvent fractions (hexane, DCM, ethyl acetate and butanol) and isolated compounds obtained from the leaves were evaluated for their inhibitory effects on selected bacteria, a fungus (Candida albicans), cyclooxygenase (COX-2), α-glucosidase and acetylcholinesterase (AChE). Amongst the compounds, quercetin (3) was the most active against Bacillus subtilis and Candida albicans while myricetrin-3-O-rhamnoside (1) and trans-N-(p-coumaroyl) serotonin (4) were the most active compounds against Escherichia coli, Klebsiella pneumonia and Staphylococcus aureus. The inhibitory activity of myricetrin-3-O-rhamnoside (1) against COX-2 was insignificant while that of the other three compounds 2–4 was low. The AChE inhibitory activity of the alkaloid, trans-N-(p-coumaroyl) serotonin was high, with a percentage inhibitory activity of 72.6% and an IC50 value of 15.0 µg/mL. The rest of the compounds only had moderate activity. Croton menyharthii leaf extracts and isolated compounds inhibit α-glucosidase at very low IC50 values compared to the synthetic drug acarbose. Structure activity relationship of the isolated flavonols 1–3 is briefly outlined. Compounds 1–4 and the leaf extracts exhibited a broad spectrum of activities. This validates the ethnomedicinal use of the plant in folk medicine.

Anti-Plasmodial Activity of Some Zulu Medicinal Plants and of Some Triterpenes Isolated from Them

Mimusops caffra E. Mey. ex A.DC and Mimusops obtusifolia Lam (both members of the Sapotaceae family), and Hypoxis colchicifolia Bak (family Hypoxidaceae) are used by traditional healers in Zululand to manage malaria. Anti-plasmodial investigation of the crude extracts and some triterpenes isolated from the plants showed activity against a chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). Among the crude extracts the leaves of M. caffra exhibited the highest activity, with an IC50 of 2.14 μg/mL. The pentacyclic tritepenoid ursolic acid (1), isolated from the leaves of M. caffra was the most active compound (IC50 6.8 μg/mL) as compared to taraxerol (2) and sawamilletin (3) isolated from the stem bark of M. obtusifolia (IC50 > 100). Chemical modification of the ursolic acid (1) to 3β-acetylursolic acid (4) greatly enhanced its anti-plasmodial activity. Compound 4 reduced parasitaemia against Plasmodium berghei by 94.01% in in vivo studies in mice. The cytotoxicity of 3β-acetylursolic acid (IC50) to two human cell lines (HEK293 and HepG2) was 366.00 μg/mL and 566.09 μg/mL, respectively. The results validate the use of these plants in folk medicine.

Study on Gas Chromatography–Mass Spectrometry Fingerprint of Acanthopanax brachypus

As a peculiar folk medicinal plant, Acanthopanax brachypus was widely used to treat various diseases in China. At present, however, there is not a good quality standard for its quality evaluation. In this study, on the basis of the validation tests of precision, stability and repeatability, the chromatographic fingerprint of A. brachypus was established by using gas chromatography (GC)-flame ionization detector (FID) and GC-MS techniques, as well as computer aided similarity evaluation system. Thirty-two different batches of samples collected from the different producing regions and the different parts of A. brachypus were studied. The results showed that the dominant constituents of all oils were monoterpenes and sesquiterpenes, as well as oxygenated monoterpenes and sesquiterpenes. The fingerprinting profiles were found to be consistent for the fresh stem bark acquired from various production areas, 48 common peaks were determined, but the relative abundance of peaks was varied. β-Pinene, linalool, p-cymene, spathulenol, camphene, endo-borneol, verbenone, β-caryophyllene, γ-terpinene, germacrene-D, camphor, β-thujone and β-cadinene were the main constituents of the fresh stem bark oil. Except for the leaf, the chemical components among different medicinal parts of fresh plant were inconsistent with the stem bark. Besides, the varieties and relative levels of chemical components in the fresh stem bark were more abundant than in the dry counterpart. The GC-MS fingerprint can be successfully applied to distinguish the substitute or adulterant, and further assess the differences of A. brachypus grown in various areas of China.

Antiplasmodial activity of some plants used by Zulu traditional healers and some of triterpenes isolated from the plants

Mimusops caffra E. Mey. ex A.DC., Mimusops obtusifolia, Lam (both members of Sapotaceae family) and Hpoxis colchicifolia, Bak are used by traditional healers in Zululand to manage malaria. We have investigated the anti-plasmodial activity of the crude extracts and isolated pure compounds of these plants. The crude dichloromethane extracts of the plants showed activity against the chloroquine sensitive (CQS) strain of Plasmodium falciparum (D10). The crude plants extracts exhibited activity with IC50 values raging from 2.14 µg/ml to 32.5 µg/ml. The pentacyclic triterpenoids (PTCs) isolated and characterized (through IR, NMR, MS spectral data) from the leaves and stem bark of M. caffra and M. obtusifolia include taraxerol (I), Sawamilletin (II), and ursolic acid (III). Ursolic acid was found to have anti-plasmodial activity (IC50, 6.8 µg/ml). The results validate the use of these plants in folk medicine.

An ethnobotanical study of medicinal plants in Turgutlu (Manisa—Turkey)

Ethnopharmacological relevanceA comprehensive ethnobotanical study was made in Turgutlu. This paper contains folk medicinal plants and ethnopharmacological information of this ethnobotanical study. Aim of studyThe aim of this study is to collect and identify the plants used in therapy by the local people and to reveal information on traditional herbal medicine. Materials and methodsThis study was made between 2010 and 2011 and its materials were the plants collected during the field work. The information was obtained through open and semi-structured interviews with the local people. In addition, informant consensus factor (FIC) and use value (UV) were calculated. Results76 Folk medicinal plants belonging to 44 families were identified in this study. Among them, 68 species are wild and eight species are cultivated plants. The most common families are Asteraceae (11.8%), Rosaceae (9.2%), Lamiaceae (7.9%), Apiaceae (3.9%) and Malvaceae (3.9%); the most common preparations were infusion (37%) and decoction (18.5%). A total of 177 medicinal uses (remedies) was recorded. Digestive system disorders have the highest FIC (0.73). According to use value (UV) the most important plants were Rosa canina (0.75), Ficus carica subsp. carica (0.74), Tilia platyphyllos (0.71) and Vitex agnus-castus (0.70). ConclusionIn the research area the use of traditional folk medicine is still prevalent in the community especially in the villages.

Topical application of serine proteases from Wrightia tinctoria R. Br. (Apocyanaceae) latex augments healing of experimentally induced excision wound in mice

Ethnopharmacological relevanceWrightia tinctoria R. Br. (Apocyanaceae) is a folk medicinal plant known to have immunomodulatory, anti-inflammatory and antihemorrhagic potential. Wrightia tinctoria latex is used for treatment of various clinical conditions including psoriasis, blisters, mouth ulcers, and extensively for topical application on fresh wounds to promote accelerated healing. Aims of the studyTo investigate the wound healing potential of Wrightia tinctoria latex proteases using a mouse model. Materials and methodsProteolytic activity of Wrightia tinctoria latex proteases (WTLP) was determined on various substrates (casein, gelatin and collagen (type-I and IV)). The thermal stability and the class of proteases present in WTLP were determined using heat treatment and specific protease inhibitors, respectively. Excision wound model in mice was used to evaluate the healing potential of WTLP application (twice daily, 10mg/kg). Neosporin, a standard drug, was used for comparison. The progression of healing was monitored using physical (wound contraction), biochemical (collagen content, catalase and MMP activity) and histological examinations. ResultsWTLP contains thermostable serine proteases, which are completely inhibited by PMSF. WTLP showed strong caseinolytic, gelatinolytic and collagenolytic activity. The excision wound healing rate upon WTLP treatment was significantly higher than (>2-fold) the control group (49% vs. 18%, ⁎⁎p<0.01) on day 3 and throughout the study. PMSF pre-treated and heat denatured WTLP failed to promote wound healing. In addition, serial biochemical analysis of the granulation tissue demonstrated 1.5-fold more (2444±100 vs. 1579±121µg/100mg tissue) hydroxyproline content and 5.6-fold higher catalase activity (16.7±1.3 vs. 3±0.3 units/mg) compared to controls. Further, the enhanced collagen content and matrix metalloproteinase activity correlated with wound contraction rate following WTLP and Neosporin treatment. Histological analysis on day 9 confirmed complete epithelialization, re-establishment of skin structure and accelerated wound healing following WTLP treatment. ConclusionsThe thermostable serine proteases of Wrightia tinctoria latex are directly involved in the wound healing process. Our findings provide a biochemical basis for the role of WTLP in the enhancement of wound healing. The study supports traditional topical application of Wrightia tinctoria latex on fresh wounds to promote accelerated healing.