A new strategy for the preparation of antibody against natural glycoside: With astragaloside IV as an example.

A new strategy for the hapten design of natural glycoside and application for the preparation of antibody is reported in this work. With astragaloside IV (AGS-IV) as an example, C6"-CH2OH on a glucosyl group was selectively oxidized by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation to C6"-COOH, which was subsequently condensed with -NH2 on bovine serum albumin to get artificial antigen. Then, the successful preparation of artificial antigen was verified by TCL, SDS-PAGE, UV, and MALDI-TOF-MS. Finally, rabbits were immunized with artificial antigen to obtain an antibody against AGS-IV. After tests of the titer, IC50, and cross-reactivity, the results showed that the antibody prepared by TEMPO oxidation in this work had higher specificity than that the antibody prepared by conventional sodium periodate (NaIO4) oxidation. The hapten, as a carboxylic acid derivative of AGS-IV, has better water solubility than AGS IV, which is more suitable for the synthesis of the hapten-carrier protein conjugate in aqueous phase, achieving another virtue of TEMPO oxidation over NaIO4 oxidation. This new strategy provides new ideas for the design of haptens of other natural glycosides, as well as the preparation of their antibodies.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Astragaloside IV attenuates allergic inflammation by regulation Th1/Th2 cytokine and enhancement CD4(+)CD25(+)Foxp3 T cells in ovalbumin-induced asthma.

Astragaloside IV is the chief ingredient of Radix Astragali, which has been used in the Traditional Chinese Medicine as a major component of many polyherbal formulations for the repair and regeneration of injured organ and tissues. We tested the anti-asthmatic effects of AST IV and the possible mechanisms. BALB/c mice that were sensitized and challenged to ovalbumin (OVA) were treated with AST IV (40mg/kg and 20mg/kg) 1h before they were challenged with OVA. Our study demonstrated that AST IV inhibited OVA-induced increases in eosinophil count; interleukin (IL)-4 level were recovered in bronchoalveolar lavage fluid increased IFN-γ and IL-10 levels in bronchoalveolar lavage fluid. Histological studies demonstrated that AST IV substantially inhibited OVA-induced eosinophilia in lung tissue. Flow cytometry studies demonstrated that AST IV substantially increased CD4(+)CD25(+)Foxp3 T cells (Treg). Furthermore quantitative real-time (qPCR) studies demonstrated that AST IV substantially enhanced Foxp3 mRNA expression in lung tissue. These findings suggest that AST IV may effectively ameliorate the progression of airway inflammation and could be used as a therapy for patients with allergic inflammation.

Dammarane triterpenes from the leaves of Panax ginseng enhance cellular immunity.

In our search for immune stimulating materials from natural source, bioassay-guided fractionation of a methanol extract of Panax ginseng leaves led to the isolation of three dammarane triterpenes (1-3), including two previously unknown compounds 27-demethyl-(E,E)-20(22),23-dien-3ß,6α,12ß-trihydroxydammar-25-one (1) and 3ß,20(S)-dihydroxydammar-24-en-12ß,23ß-epoxy-20-O-ß-D-glucopyranoside (2). Their structures were elucidated on the basis of spectroscopic methods, chemical transformation, and by the comparison with those of literature data. Compounds 1-3 significantly increased interleukin-12 expression in LPS-activated mouse peritoneal macrophage at a concentration of 100 ng/mL. Furthermore, compound 1 strongly increased the Th1 response-mediated cytokine IL-2, and decreased Th2 response-mediated cytokines IL-4 and IL-6 expression at 100 ng/mL on ConA-activated splenocytes. This study indicated that compound 1 showed a better effect on cellular immunity, and provided new chemical entities as promising lead compounds for the treatment of cellular immunity-related diseases.

Production of polyclonal antibody against madecassoside and development of immunoassay methods for analysis of triterpene glycosides in Centella asiatica.

INTRODUCTION: Centella asiatica (L.) Urban consists of two major triterpene glycosides, asiaticoside (AS) and madecassoside (MA), as active components used for wound healing and enhancing memory. OBJECTIVE: To produce a polyclonal antibody against madecassoside (MA-PAb) and develop enzyme-linked immunosorbent assay (ELISA) and Eastern blotting methods for quantitative analysis of triterpene glycosides in Centella asiatica. METHODS: An ELISA method was developed using polyclonal antibody against MA. An Eastern blotting method on the PES membrane was established for determination of MA and AS. The immunoassays were validated for sensitivity, precision, specificity and accuracy. RESULTS: The prepared MA-PAb shows specificity to MA and AS. The measuring range of triterpene glycosides was 0.39-50 µg/mL using the ELISA method. An Eastern blotting method was developed for determining individual MA and AS, which could be detected in the range of 62.5-500 ng. The limit of detection for MA and AS was 31.25 ng. The two methods developed showed good specificity, precision, and accuracy, and also correlated with high-performance liquid chromatography. CONCLUSION: These immunoassays have several advantages that include high sensitivity as well as being rapid and facile for determination of the triterpene glycosides in C. asiatica.

Eastern blotting analysis and isolation of two new dammarane-type saponins from American ginseng.

Ginsenosides, the major active component of American ginseng, were analyzed using eastern blotting with anti-ginsenoside Rb(1) and Rg(1) monoclonal antibodies (MAbs). Immunoassay-guided fractionation of the methanol extract of American ginseng and column chromatography led to the isolation of two new minor dammarane-type saponins, named quinquenosides Ja (1) and Jb (2). Their structures were elucidated to be 6-O-[α-L-rhamnopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-[ß-D-glucopyranosyl(1→4)-ß-D-glucopyranosyl]-3ß,6α,12ß,20ß-tetrahydroxydammar-24-ene (1) and 3-O-[ß-D-glucopyranosyl(1→2)-ß-D-glucopyranosyl]-20-O-{[α-L-arabinofuranosyl(1→6)-ß-D-glucopyranosyl](1→6)-ß-D-glucopyranosyl}-3ß,12ß,20ß-trihydroxydammar-24-ene (2) on the basis of chemical and spectroscopic methods.

QSAR, docking and in vivo studies for immunomodulatory activity of isolated triterpenoids from Eucalyptus tereticornis and Gentiana kurroo.

Two triterpenoids ursolic acid (1) and lupeol (2) isolated and characterized from Eucalyptus tereticornis and Gentiana kurroo were subjected to in silico QSAR modeling and docking studies and later the predicted results were confirmed through in vivo experiments. QSAR modeling results showed that both the triterpenoids possess immunomodulatory and anti-inflammatory activity comparable to boswellic and cichoric acids, but were less active than levamisol. Docking results suggested that both the triterpenoids (1 and 2) showed immune modulatory and anti-inflammatory activity due to high binding affinity to human receptors viz., NF-kappaB p52 (-50.549 kcal/mol), tumor necrosis factor (TNF-alpha) (-47.632 kcal/mol), nuclear factor NF-Kappa-B P50 (-16.798 kcal/mol) and cyclooxygenase-2 (-55.244 kcal/mol). Further both the triterpenoids (1 and 2) were subjected to in vivo immunomodulatory activity in female Swiss albino mice. The experimental mice were divided into nine groups, each comprised of six mice. These received oral treatment for a period of 28 days. The triterpenoids (1 and 2) showed significant increased in humoral immune function, but no significant changes were observed in cell mediated immune response and hematological parameters. The in silico and in vivo experimental data suggested that both the triterpenoids 1 and 2 may be considered as potential immunomodulatory drug-like molecules.

Triterpene acids from rose hip powder inhibit self-antigen- and LPS-induced cytokine production and CD4⁺ T-cell proliferation in human mononuclear cell cultures.

A triterpene acid mixture consisting of oleanolic, ursolic and betulinic acid isolated from a standardized rose hip powder (Rosa canina L.) has been shown to inhibit interleukin (IL)-6 release from Mono Mac 6 cells. The present study examined the effects of the triterpene acid mixture on the cytokine production and proliferation of CD4⁺ T cells and CD19⁺ B cells induced by a self-antigen, human thyroglobulin and by lipopolysaccharide in cultures of normal mononuclear cells. The triterpene acid mixture inhibited the production of tumor necrosis factor-α and IL-6 with estimated IC50 values in the range 35-56 µg/mL, the Th1 cytokines interferon-γ and IL-2 (IC50 values 10-20 µg/mL) and the antiinflammatory cytokine IL-10 (IC50 values 18-21 µg/mL). Moreover, the mixture also inhibited CD4⁺ T-cell and CD19⁺ B-cell proliferation (IC50 value 22 and 12 µg/mL, respectively). Together, these data demonstrate that oleanolic, ursolic and betulinic acid are active immunomodulatory constituents of the standardized rose hip powder. However, since the estimated IC50 values are in the µg/mL range, it is questionable whether the content of the triterpene acids in the standardized rose hip powder, alone, can explain the reported clinical effects.

An enzyme-linked immunosorbant assay using monoclonal antibody against bacoside A3 for determination of jujubogenin glycosides in Bacopa monnieri (L.) Wettst.

INTRODUCTION: In Ayurvedic medicines, Bacopa monnieri (L.) Wettst. (brahmi) is known as a medicinal plant used for memory enhancement. Its active compounds are classified as pseudojujubogenin and jujubogenin glycosides. Owing to the lack of chromophore in the saponin glycoside structures, HPLC-UV-vis gives low sensitivity for determination of such compounds. In the case of the detection of small amounts of saponin glycosides, immunological assay could be a suitable method. OBJECTIVE: To develop and validate a sensitive enzyme-linked immunosorbant assay (ELISA) using monoclonal antibody (MAb) against bacoside A3, the major jujubogenin glycoside found in brahmi. METHODOLOGY: An immunogen was prepared by conjugating bacoside A3 with a bovine serum albumin (BSA). To determine its immunogenicity, the ratio of hapten in bacoside A3-BSA conjugate was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). After immunisation in mice, hybridomas secreting MAbs against bacoside A3 were produced by fusing the immunised splenocytes with SP2/0- Ag14 myeloma cells. The antibody was raised specifically against jujubogenin glycosides. The ELISA using anti-bacoside A3 MAb was developed. RESULTS: Bacoside A3 in the range of 3.05-97.70 ng mL⁻¹ could be detected by ELISA using anti-bacoside A3 MAb. The assay showed a detection limit of 0.48 ng mL⁻¹ (0.517 nm). The validation study showed that the method was precise, accurate and sensitive. Interestingly, the MAb showed cross-reactivity with the other jujubogenin glycosides, bacopaside X and IV. However, it did not show cross-reactivity with any of pseudojujubogenin glycosides. CONCLUSION: The study demonstrated that ELISA using anti-bacoside A3 MAb can be used for determination of total jujubogenin glycosides in brahmi.

The known immunologically active components of Astragalus account for only a small proportion of the immunological adjuvant activity when combined with conjugate vaccines.

The 95 % ethanol extract of Astragalus has been demonstrated to have potent activity as an immunological adjuvant when administered with vaccines of various types. We endeavor here to identify the components of this extract that are responsible for this adjuvant activity. Mice were immunized with KLH conjugated to cancer carbohydrate antigens globo H and GD3 and cancer peptide antigen MUC1 combined with different Astragalus fractions or with commercially available Astragalus saponins and flavonoids. The antibody responses against cancer antigens and KLH were quantitated in ELISA assays, and toxicity was calculated by weight loss. Astragalosides II and IV were the most active components, but the toxicity of these two differed dramatically. Astragaloside II was the most toxic Astragalus component with 5-10 % weight loss at a dose of 500 µg while astragaloside IV showed no weight loss at all at this dose, suggesting that astragaloside IV might be utilized as an immunological adjuvant in future studies. Several flavonoids also had significant adjuvant activity. However, when the activities of these known immunologically active components of Astragalus (and of endotoxin) are calculated based on the extent of their presence in the 95 % ethanol extract, they provide only a small proportion of the immunological activity. This raises the possibility that additional uniquely active components of Astragalus may contribute to adjuvant activity, or that the adjuvant activity of Astragalus is greater than the activity of the sum of its parts.

Preparation of a specific monoclonal antibody to asiaticoside for the development of an enzyme-linked immunosorbent assay.

Asiaticoside (AS), the major active component of Centella asiatica (L.) Urban, is used as a memory enhancer and for wound healing. We have successfully prepared monoclonal antibodies (MAbs) against AS, and developed an enzyme-linked immunosorbent assay (ELISA) system for its determination. AS was conjugated to the carrier protein bovine serum albumin (BSA), which acted as an immunogen. In order to confirm its immunogenicity, the ratio of hapten in the AS-BSA conjugate was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). After immunization, hybridomas secreting MAbs against AS were produced by fusing splenocytes with the mouse myeloma cell line, SP2/0-Ag14. After the screening, anti-asiaticoside MAb 2B4 was obtained. Weak cross-reactivities occurred with madecassoside (7.08%), but no cross-reactivities were observed with other related triterpenoid glycosides (<0.01%). The assay was suitable for quantitating AS in the range of 0.78 to 50 µg mL(-1). A good correlation of AS concentrations in crude extracts of C. asiatica between ELISA and HPLC methods was obtained (r(2) = 0.999). The contents of AS in various cultivated C. asiatica samples were assayed by the newly established ELISA. The recovery rates of AS in the samples were in the range of 95-103% with coefficients of variation of <10%. The intra- and inter-assay variations were 3.9 and 4.5%, respectively. The ELISA method described should prove useful as an analytical tool for quality control and standardization of medicinal plants and pharmaceutical products containing AS.

Immunochromatographic assay for the detection of pseudojujubogenin glycosides.

INTRODUCTION: Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE: The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY: The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS: The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION: The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.

Determination of pseudojujubogenin glycosides from Brahmi based on immunoassay using a monoclonal antibody against bacopaside I.

Bacopa monnieri (L.) Wettst. (Brahmi) is an Ayurvedic medicinal plant used for centuries as a memory enhancer. Dammarane-type triterpenoid saponins classified as pseudojujubogenin and jujubogenin glycosides are reported as the active components in this plant. In this study, the monoclonal antibody (MAb) against bacopaside I, a major pseudojujubogenin glycoside found in Brahmi, was produced and characterised. Bacopaside I was conjugated with bovine albumin serum to prepare an immunogen. Hybridomas secreting a monoclonal antibody against bacopaside I was produced by fusing splenocytes from an immunised mouse and SP2/0-Ag14 myeloma cells. The results showed that the antibodies were raised specifically against pseudojujubonin glycosides. An enzyme-linked immunosorbant assay (ELISA) using anti-bacopaside I MAb was performed in the range of 1.95-62.5 ng/mL of bacopaside I, with a limit of detection of 0.5 ng/mL. The method was validated and the practical use of the ELISA for analysing saponin glycosides from Brahmi was demonstrated.

An enzyme-linked immunosorbant assay using polyclonal antibodies against bacopaside I.

Bacopa monnieri (L.) Wettst. (Brahmi) is a medicinal plant used as a memory enhancer in Ayurvedic medicines. Its active components are triterpenoid glycosides namely pseudojujubogenin and jujubogenin glycosides. In order to analyze these saponin glycosides, an enzyme-linked immunosorbant assay (ELISA) was developed using polyclonal antibodies against bacopaside I, one of the pseudojujubogenin glycosides found in the plant. Bacopaside I was conjugated with a bovine albumin serum (BSA) to prepare an immunogen. The bacopaside I-BSA conjugate was immunized to a rabbit for producing polyclonal antibodies (PAbs). The results showed that the antibodies were raised specifically against pseudojujubogenin glycosides. An ELISA using anti-bacopaside I PAbs was performed in the range of 1.95-62.5 ng mL(-1) of bacopaside I and the limit of detection was 0.1 ng mL(-1). The method was validated and the applicability of the ELISA for analyzing saponin glycosides from Brahmi was demonstrated.

Adjuvant effects of protopanaxadiol and protopanaxatriol saponins from ginseng roots on the immune responses to ovalbumin in mice.

Protopanaxadiol saponins (Rg3, Rd, Rc, Rb1 and Rb2) and protopanaxatriol saponins (Rg1, Re and Rg2) isolated from the root of Panax ginseng C.A. Meyer were evaluated for their adjuvant effects on the immune responses to ovalbumin (OVA) in mice. BALB/c mice were subcutaneously injected twice at a 3-week interval with 10 microg of ovalbumin or 10 microg of OVA plus 50 microg of ginsenosides Rg3, Rd, Rc, Rb1, Rb2, Rg1, Re or Rg2 or Quil A (n=5). Blood samples were collected for measuring specific total-IgG, IgG1 and IgG2a, and splenocytes were harvested for determining lymphocyte proliferation as well as IFN-gamma and IL-5 production 2 weeks after the boosting. The results indicated that OVA-specific antibody responses were significantly higher in mice immunized with OVA co-administered with Rg1, Re, Rg2, Rg3 and Rb1 but not with Rd, Rc and Rb2 when compared with the control (immunized with OVA only). Significantly enhanced splenocyte proliferative responses to Con A, LPS and OVA as well as the production of both IL-5 and IFN-gamma stimulated by OVA were also detected in mice immunized with OVA co-administered with Rg1 but not with Rb1, Re and Rg3. Of the ginsenosides studied, Rg1, Re, Rg2, Rg3 and Rb1 have more potent adjuvant properties than the others, indicating that they are the major constituents contributing to the adjuvant activities of total ginseng saponins. Varieties of ginsenosides in adjuvant activity might be attributed to the varieties of molecular conformations determined by the side sugar chains attaching to their dammarane skeleton.

Immunoaffinity chromatography for the sample pretreatment of Taxus plant and cell extracts prior to analysis of taxanes by high-performance liquid chromatography.

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

Structurally related immunological effects of triterpenoid saponins.

The influence of the triterpenoid saponins 1-10 has been investigated on murine spleenocytes in the lymphocyte transformation test and on murine macrophages in an phagocytosis assay. The lymphocyte transformation test and the phagocytosis assay showed that the tested compounds have no stimulating effect. However, a significant inhibition of lymphocyte proliferation by the triterpenoid saponins 2, 6 and 10 was demonstrated.

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis-tetanus vaccines.

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 microg) was intranasally (i.n.) inoculated with influenza vaccine (10 microg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3-14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 microg) was i.n. inoculated with the vaccine (10 microg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27-50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 microg) and influenza HA vaccine (1 microg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 microg) each and diphtheria-pertussis-tetanus (DPT) vaccine (10 microg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 microg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.

Immunological detection and quantitation of 10-deacetylbaccatin III in Taxus sp. plant and tissue cultures.

A high-sensitive ELISA method was developed for the detection and semi-quantitative determination of 10-deacetylbaccatin III and its structurally related compounds in crude extract of Taxus sp. plants and tissue cultures. The antibodies were raised in rabbits using 7- or 10-succinyl-10-deacetylbaccatin III-BSA conjugate as immunogen. The working range of the assay was from 0.003 to 1.000 ng (0.09 to 31.33 nM) of 10-deacetylbaccatin III per assay. The cross-reacting material in crude plant extract was examined by chromatographic (silica gel CC, HPLC) and immunoassay methods. Study on the evaluation of cross-reacting material in crude Taxus plant extracts showed that at least 80% of the immunosignal correspond to 10-deacetylbaccatin III in the extract. The ELISA method was applied to investigate the 10-deacetylbaccatin III equivalent content in crude extracts of 19 plants species including Taxaceae, Taxodiaceae and Pinaceae species. The 10-deacetylbaccatin III-like structure was only detected in Taxus and Torreya sp. The results indicate that this immunoassay is a useful tool for the rapid screening of species, varieties or individual plants out of a wide population. The distribution of 10-deacetylbaccatin III equivalent content in 9-month old Taxus plantlets cultivated in vitro as well as in callus culture was investigated.

Centella asiatica (Indian pennywort), an effective therapeutic but a weak sensitizer.

The sensitizing capacity of Centella asiatica (raw extract) and its triterpenic constituents asiaticoside, asiatic acid and madecassic acid has been studied in guinea pigs. The extract itself as well as the 3 acids were found to be very weak sensitizers. Centella asiatica extract is used effectively in the treatment of keloids, leg ulcers, phlebitis, slow-healing wounds, leprosy, surgical lesions, striae distensae and cellulitis. Although applied frequently to damaged skin, the risk of acquiring contact sensitivity to this plant or its constituents is low.