Periploca Sepium Bge Research Articles

Discovery of potential Q-marker of traditional Chinese medicine based on plant metabolomics and network pharmacology: Periplocae Cortex as an example

BackgroundQuality control exerted great importance on the clinical application of drugs for ensuring effectiveness and safety. Due to chemical complexity, diversity among different producing areas and harvest seasons, as well as unintentionally mixed with non-medicinal parts, the current quality standards of traditional Chinese medicine (TCM) still faced challenges in evaluating the overall chemical consistency. PurposeWe aimed to develop a new strategy to discover potential quality marker (Q-marker) of TCM by integrating plant metabolomics and network pharmacology, using Periplocae Cortex (GP, the dried root bark of Periploca sepium Bge.) as an example. MethodsFirst, plant metabolomics analysis was performed by UPLC/Q-TOF MS in 89 batches of samples to discover chemical markers to distinguish medicinal parts (GP) and non-medicinal parts (the dried stem bark of Periploca sepium Bge. (JP)), harvest seasons and producing region of Periplocae Cortex. Second, network pharmacology was applied to explore the initial linkages among chemical constituents, targets and diseases. Last, potential Q-marker were selected by integrating analysis of plant metabolomics and network pharmacology, and the quantification method of Q-marker was developed by using UPLC-TQ-MS. ResultsThe chemical profiling of GP and JP was investigated. Fifteen distinguishing features were designated as core chemical markers to distinguish GP and JP. Besides, the content of 4-methoxybenzaldehyde-2-O-β-d-xylopyranosyl-(1→6)-β-d-glucopyranoside could be used to identify Periplocae Cortex harvested in spring-autumn or summer. Meanwhile, a total of 15 components targeted rheumatoid arthritis were screened out based on network pharmacology. Taking absorbed constituents into consideration, 23 constituents were selected as potential Q-marker. A simultaneous quantification method (together with 11 semi-quantitative analysis) was developed and applied to the analysis of 20 batches of commercial Periplocae Cortex on the market. The PLS-DA model was successfully developed to distinguish GP and JP samples. In addition, the artificially mixed GP sample, which contained no less than 10% of the adulterant (JP), could also be correctly identified. ConclusionOur results indicated that 9 ingredients could be considered as Q-marker of Periplocae Cortex. This study has also demonstrated that the plant metabolomics and network pharmacology could be used as an effective approach for discovering Q-marker of TCM to fulfill the evaluation of overall chemical consistency among samples from different producing areas, harvest seasons, and even those commercial crude drugs, which might be mixed with a small amount of non-medicinal parts.

Combination of the natural compound Periplocin and TRAIL induce esophageal squamous cell carcinoma apoptosis in vitro and in vivo: Implication in anticancer therapy

BackgroundEsophageal cancer is one of the most common malignant tumors in the world. With currently available therapies, only 20% ~ 30% patients can survive this disease for more than 5 years. TRAIL, a natural ligand for death receptors that can induce the apoptosis of cancer cells, has been explored as a therapeutic agent for cancers, but it has been reported that many cancer cells are resistant to TRAIL, limiting the potential clinical use of TRAIL as a cancer therapy. Meanwhile, Periplocin (CPP), a natural compound from dry root of Periploca sepium Bge, has been studied for its anti-cancer activity in a variety of cancers. It is not clear whether CPP and TRAIL can have activity on esophageal squamous cell carcinoma (ESCC) cells, or whether the combination of these two agents can have synergistic activity.MethodsWe used MTS assay, flow cytometry and TUNEL assay to detect the effects of CPP alone or in combination with TRAIL on ESCC cells. The mechanism of CPP enhances the activity of TRAIL was analyzed by western blot, dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP) assay. The anti-tumor effects and the potential toxic side effects of CPP alone or in combination with TRAIL were also evaluated in vivo.ResultsIn our studies, we found that CPP alone or in combination with TRAIL could inhibit the proliferation of ESCC cells and induce apoptosis, and we certificated that combination of two agents exert synergized functions. For the first time, we identified FoxP3 as a key transcriptional repressor for both DR4 and DR5. By down-regulating FoxP3, CPP increases the expression of DR4/DR5 and renders ESCC cells much more sensitive to TRAIL. We also showed that CPP reduced the expression of Survivin by inhibiting the activity of Wnt/β-catenin pathway. All these contributed to synergistic activity of CPP and TRAIL on ESCC cells in vitro and in vivo.ConclusionOur data suggest that CPP and TRAIL could be further explored as potential therapeutic approach for esophageal cancer.

Periplocin Sensitizes Human Esophageal Squamous Cell Carcinoma to Trail-Induced Apoptosis via Upregulating Death Receptors and Inhibiting the Wnt/β-Catenin Pathway

Background: Esophageal cancer is one of the most common malignant tumors in the world. With currently available therapies, only 20% ~30% patients can survive this disease for more than 5 years. Although TRAIL, a natural ligand for death receptors that can induce the apoptosis of cancer cells, has been explored as a therapeutic agent for cancers, it has been reported that many cancer cells are resistant to TRAIL, limiting the potential clinical use of TRAIL as a cancer therapy. Meanwhile, Periplocin (CPP), a natural compound from dry root of Periploca sepium Bge, has been studied for its anti-cancer activity. It is not clear if CPP and TRAIL can have activity on esophageal squamous cell carcinoma (ESCC) cells, or if the combination of these two agents can have synergistic activity. Methods: We used MTS assay, flow cytometry and TUNEL assay to detect the effects of CPP alone and in combination with TRAIL in ESCC cells. The mechanism of CPP enhances the activity of TRAIL was analyzed by western blot, dual luciferase reporter gene assay and ChIP assay and so on. The anti-tumor effects and the potential toxic side effects of CPP alone and in combination with TRAIL were also evaluated in vivo. Findings: In our studies, we found that CPP alone and in combination with TRAIL could inhibit the proliferation ESCC cells and induce apoptosis. For the first time, we identified FoxP3 as a key transcriptional repressor for both DR4 and DR5. By down-regulating FoxP3, CPP increases the expression of DR4/DR5 and renders ESCC cells much more sensitive to TRAIL. We also showed that CPP reduced the expression of Survivin by inhibiting the activity of Wnt/β-catenin pathway. All these contribute to synergistic in vitro and in vivo activity of CPP and TRAIL on ESCC cells. Interpretation: Our data suggests that CPP and TRAIL can be further explored as potential therapeutic approach for esophageal cancer. Funding: This project was supported by the Natural Science Foundation of China (Grant No. 81673642, 81772550). Conflict of Interest: The authors declared no conflict of interest. Ethical Approval: All animal experiments were approved by the Animal Care Committee of the Fourth Hospital of Hebei Medical University.

Effects of an immunosuppressive active component of Periplocae Cortex (Periploca sepium Bge.) on positive selection of thymocytes in vitro

Abstract Objective To determine the effect of an immunosuppressive active component (periploside A) isolated from the stem bark of Periplocae Cortex (Periploca sepium Bge.), a Chinese medicinal herb used in the treatment of rheumatoid arthritis for centuries in China, on positive selection of thymocytes in vitro. Methods Female C57BL/6 mice at 6 weeks of age were housed in specific pathogen-free conditions. Double-positive thymocytes from C57BL/6 mice were induced into positive selection in vitro with or without periploside A treatment. Cell viability and expression of CD69, CD4, and CD8 were analyzed by flow cytometry. Results Flow cytometric examination of thymocyte populations revealed that the percentage of CD8+ single-positive thymocytes was decreased by periploside A upon differentiation induced by an anti-CD3 antibody. However, the percentage of CD4+ single-positive thymocytes was decreased by periploside A upon differentiation induced by phorbol 12-myristate 13-acetate/ionomycin. Expression of CD69 plays a major role in prohibiting differentiation of thymocytes. Treatment with periploside A decreased CD69 expression in thymocytes. Conclusion These results demonstrate that periploside A influences positive selection of thymocytes in vitro.

Periplocoside A ameliorated type II collagen-induced arthritis in mice via regulation of the balance of Th17/Treg cells

Periplocoside A (PSA) has been extracted from the Chinese herbal medicine Periploca sepium Bge to treat rheumatoid arthritis (RA) via immune regulation. We previously found that PSA exhibits immunosuppressive activity both in vitro and in vivo. Balanced regulation of helper T 17 (Th17)/regulatory T (Treg) cells is the current therapeutic direction for the treatment of RA. The present study investigated the mechanism of PSA in treating collagen-induced arthritis (CIA). The therapeutic effects and potential pharmacological mechanisms of PSA were specifically clarified by examining its effects on CIA in DBA/1 mice. PSA administration significantly relieved the severity of the arthritis, and preventive administration of PSA reduced the incidence of arthritis in the mice with CIA and relieved joint damage in terms of morphology. PSA was also able to reduce the levels of anti-collagen II (CII) antibodies and pro-inflammatory cytokines in the serum. As a result, the proportion of Th17 cells decreased, and the proportion of Treg cells increased. A follow-up study of the ex vivo immunological reactions induced by a specific antigen found that PSA suppressed lymphocyte proliferation, inhibited the differentiation and reactivity of Th17 cells, and promoted the proportion of Treg cells among helper T cells. PSA also exhibited pharmacological effect in regulating the balance between Th17 and Treg cells in CIA through relevant signalling pathways. Thus, PSA played a specific role in CIA treatment. In particular, our results suggest that the therapeutic effects of PSA on RA are partially realized via the regulation of the balance of Th17/Treg cells.

Validation of biomarkers in cardiotoxicity induced by Periplocin on neonatal rat cardiomyocytes using UPLC-Q-TOF/MS combined with a support vector machine

Corex Periplocae (the root of Periploca sepium Bge) has been widely used in clinics. Periplocin, as one of the components of cardiac glycosides in Corex Periplocae, easily triggers cardiotoxicity when used improperly. To evaluate the toxicity of Periplocin, we used UPLC-Q-TOF/MS to investigate metabolic profiles on neonatal rat cardiomyocytes exposed to high and low doses of Periplocin (0.2mmol/L, 0.4mmol/L). Finally, we identified 11 biomarkers associated with toxicity through multivariate statistical analysis. A “supervised” Support Vector Machine (SVM) study was used to optimize and verify the reliability of these biomarkers. In these biomarkers, all biomarkers, including carnitine, acetylcarnitine, lysoPC(16:0), proline, glutamic acid, pyroglutamic acid, leucine, pantothenic acid, tryptophan, indoleacrylic acid and citric acid, revealed a downward trend with the increase of dosage. Moreover, pathway analysis showed that these metabolites were associated with amino acid metabolism, energy metabolism and sphingolipid metabolism, which contributes to a further understanding of the toxicity mechanism of Corex Periplocae and its clinical safety. Additionally, we demonstrate that an UPLC-Q-TOF/MS-based metabolomic approach is a powerful tool and provides a promising approach for assessing the toxicity of traditional Chinese medicine and drug safety screening.

P-glycoprotein- and organic anion-transporting polypeptide-mediated transport of periplocin may lead to drug-herb/drug-drug interactions.

Periplocin, an active and toxic component of the traditional Chinese herbal medicine Periploca sepium Bge, is a cardiac glycoside compound that has been implicated in various clinical accidents. This study investigated the role of transporters in the intestinal absorption and biliary excretion of periplocin, as well as the possible metabolic mechanism of periplocin in liver S9. In a bidirectional transport assay using Madin–Darby canine kidney (MDCK) and MDCK multidrug-resistance protein (MRP)-1 cell monolayers, both in situ intestinal and liver-perfusion models were used to evaluate the role of efflux and uptake transporters on the absorption and biliary excretion of periplocin. In addition, in vitro metabolism of periplocin was investigated by incubating with human/rat liver S9 homogenate fractions to evaluate its metabolic mechanisms in liver metabolic enzymes. The results showed that P-glycoprotein (P-gp) was involved in the intestinal absorption of periplocin, whereas MRP2 and breast cancer-resistance protein were not. The efflux function of P-gp may be partly responsible for the low permeability and bioavailability of periplocin. Moreover, both inhibitors of P-gp and organic anion-transporting polypeptides (OATPs) increased periplocin biliary excretion. No obvious indications of metabolism were observed in the in vitro incubation system, which suggests that periplocin did not interact with the hepatic drug metabolic enzymes. The results of this study showed that the efflux and uptake transporters P-gp and OATPs were involved in the absorption and biliary excretion of periplocin, which may partially account for its low permeability and bioavailability. As a toxic compound, potential drug–herb/herb–herb interactions based on OATPs and P-gp should be taken into account when using P. sepium Bge in the clinic.

Lack of Mutagenicity Potential of <i>Periploca sepium</i> Bge. in Bacterial Reverse Mutation (Ames) Test, Chromosomal Aberration and Micronucleus Test in Mice

ObjectivesThe root barks of Periploca sepium Bge. (P. sepium) has been used in traditional Chinese medicine for healing wounds and treating rheumatoid arthritis. However, toxicity in high-doses was often diagnosed by the presence of many glycosides. The potential mutagenicity of P. sepium was investigated both in vitro and in vivo.MethodsThis was examined by the bacterial reverse mutation (Ames) test using Escherichia coli WP2uvrA and Salmonella typhimurium strains, such as TA98, TA100, TA1535, and TA1537. Chromosomal aberrations were investigated using Chinese hamster lung cells, and the micronucleus test using mice.ResultsP. sepium did not induce mutagenicity in the bacterial test or chromosomal aberrations in Chinese hamster lung cells, although metabolic activation and micronucleated polychromatic erythrocytes were seen in the mice bone marrow cells.ConclusionsConsidering these results, it is suggested that P. sepium does not have mutagenic potential under the conditions examined in each study.

Periplocoside A prevents experimental autoimmune encephalomyelitis by suppressing IL-17 production and inhibits differentiation of Th17 cells

The aim of this study was to determine the therapeutic effect of Periplocoside A (PSA), a natural product isolated from the traditional Chinese herbal medicine Periploca sepium Bge, in MOG(35-55) (myelin oligodendrocyte glycoprotein 35-55)-induced experimental autoimmune encephalomyelitis (EAE). Female C57BL/6 mice immunized with MOG(35-55) were treated with (50 mg/kg or 25 mg/kg) or without PSA following immunization and continuously throughout the study. The degree of CNS inflammation was evaluated by H&E staining. Anti-MOG-specific recall responses were analyzed by [3H]-Thymidine incorporation, ELISA, and RT-PCR. The proportion of IL-17-producing T cells was measured by flow cytometry. Oral administration of PSA significantly reduced the incidence and severity of EAE, which closely paralleled the inhibition of MOG(35-55)-specific IL-17 production. Importantly, PSA inhibited the transcription of IL-17 mRNA and RORgammat. Further studies examining intracellular staining and adoptive transfer EAE validated the direct suppressive effect of PSA on Th17 cells. In vitro studies also showed that PSA significantly inhibited the differentiation of Th17 cells from murine purified CD4+ T cells in a dose-dependent manner. PSA ameliorated EAE by suppressing IL-17 production and inhibited the differentiation of Th17 cells in vitro. Our results provide new insight into the potential mechanisms underlying the immunosuppressive and anti-inflammatory effects of PSA.

Immunosuppressive pregnane glycosides from Periploca sepium and Periploca forrestii

Nine pregnane glycosides containing peroxy functions in their sugar moieties ( 1– 5 and 11– 14), five oligosaccharides ( 6– 10), six pregnane glycosides ( 15– 20), and five cardiac glycosides ( 21– 25) were isolated from the root barks of Periploca sepium Bge. (Asclepiadaceae) and the roots of Periploca forrestii Schltr. (Asclepiadaceae), two traditional Chinese medicines used for the treatment of rheumatoid arthritis. Among them, 1– 8 are hitherto unknown. Their structures were characterized on the basis of spectroscopic analyses. In pharmacological testing, compounds 1– 5 and 11– 14 were found to exhibit inhibitory activity against the proliferation of T lymphocyte in vitro with IC 50 values ranging from 0.29 μM to 1.97 μM, while the other components showed no significant inhibitory activity.

Five New Pregnane Glycosides from the Root Barks of Periploca sepium

AbstractFive new pregnane glycosides, perisepiumosides A–E (1–5, resp.), along with the seven known constituents 6–12, were isolated from the root barks of Periploca sepium Bge. (Asclepiadaceae), a traditional Chinese medicine used for the treatment of rheumatoid arthritis and wounds. Their structures were characterized on the basis of spectroscopic analyses.

Effects of Iso-osmotic Potential Salt and Water Stress on the Seed Germination of Periploca sepium Bge

The seed germination rates,seeding fresh weights,daily taproot lengths,seed vigor indexes and germination value of Periploca sepium Bge.was investigated under iso-osmotic potential NaCl and PEG with different osmotic potential.The regression analyses were done between the seed germination and different osmotic potential.It was shown that(1) the total germination rates,seeding fresh weights,seed vigor indexes and seed germination value of P.sepium Bge.tended to decrease as the osmotic potential of NaCl and PEG become intensified;the seed germination was restrained more seriously by PEG than by NaCl under the high osmotic potential(≥-0.5MPa),and low osmotic potential(≤-1.4MPa) through meta-analysing seed vigor indexes and germination value,but it reversed under other ranges;(2)the daily germination rates and taproot lengths of P.sepium Bge.indicated that compared with NaCl,PEG delayed the seed germination and inhibited taproot lengths growth more seriously;(3)the regression equation between the seed germination and different osmotic potential under salt and water stress showed that the critical value of the seed of P.sepium Bge.was-0.9MPa and the maximum value was-1.3MPa under NaCl,while they were-1.0MPa and-1.4MPa under the PEG.When the osmotic stress was released,the germination recovery rates of the different osmotic potential has achieved 100%.It was resulted that P.sepium Bge.owns the characteristics of salinity tolerance and drought resistance.

Periplocoside A, a pregnane glycoside from Periploca sepium Bge, prevents concanavalin A-induced mice hepatitis through inhibiting NKT-derived inflammatory cytokine productions

Periploca sepium Bge, a traditional Chinese herb medicine, is widely used for treating rheumatoid arthritis in china. Periplocoside A (PSA), a pregnane glycoside, is a new nature product compound isolated from P. sepium Bge. We examined the protective effects of PSA, on concanavaline A (ConA)-induced hepatitis. Pretreatment with PSA dramatically ameliorated ConA-induced liver injury, which was characterized by reducing serum alanine transaminase (ALT), pathogenic cytokines of interleukin (IL)-4 and interferon (IFN)-γ levels, impeding the liver necrosis, and thus elevating the survival rate. In vitro, PSA inhibited IL-4 and IFN-γ productions of α-galactosylceramide (α-GalCer) or anti-CD3-activated Natural killer T (NKT) cells. Enzyme Linked Immunosorbent Assay (ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR) assays revealed PSA suppressed IL-4 transcription and IFN-γ translation. In conclusion, PSA had significantly preventative effect on ConA-induced hepatitis, which was closely associated with inhibition of NKT-derived inflammatory cytokine productions. These findings suggested that PSA has the therapeutic potential for treatment of human autoimmune-related hepatitis.

Periplocoside E, an Effective Compound fromPeriploca sepiumBge, Inhibited T Cell Activation in Vitro and in Vivo

Periploca sepium Bge, a traditional Chinese herb medicine, is used for treating rheumatoid arthritis in China. Followed the bioactivity-guided isolation, the most potent immunosuppressive compound, periplocoside E (PSE), a pregnane glycoside, had been identified from P. sepium Bge. We investigated the immunosuppressive effects of PSE in vitro and in vivo. The results showed that PSE in a dose-dependent manner significantly inhibited the proliferation of splenocytes induced by concanavalin A and mixed lymphocyte culture reaction at no cytotoxic concentrations (<5 microM). Administration of PSE suppressed a delayed-type hypersensitivity reaction, and ovalbumin (OVA) induced antigen-specific immune responses in mice. In vivo treatment with PSE dose dependently suppressed OVA-induced proliferation and cytokine [interleukin (IL)-2 and interferon (IFN)-gamma] production from splenocytes in vitro. Purified T cells from OVA-immunized mice with PSE treatment showed its low ability for activation by OVA plus normal antigen presenting cell stimulation again in vitro. Further studies showed PSE dose dependently inhibited anti-CD3-induced primary T cell proliferation, activation for IL-2Ralpha (CD25) expression, and cytokine (IFN-gamma and IL-2) production also at the transcriptional level. PSE was highly specific and significantly inhibited the activation of extracellular signal-regulated kinase and Jun N-terminal kinase, whereas activation of p38 was not affected in T cells stimulated with anti-CD3. These results demonstrated that PSE is an immunosuppressive compound in P. sepium Bge, which directly inhibits T cell activation in vitro and in vivo. This study provided evidence to understand the therapeutic effects of P. sepium Bge and indicated that this herb is appropriate for treatment of T cell-mediated disorders, such as autoimmune diseases.

Studies on chemical constituents of antitumor fraction from Periploca sepium BGE. I.

When the CHCl3 extract obtained by partitioning the methanolic extract of Periploca sepium between H2O and CHCl3 was subjected to column chromatography on silica gel, the antitumor activity against Sarcoma 180 ascites in mice was concentrated in the fraction eluted with CHCl3-MeOH (10 : 1). From the antitumor fraction, eight products, S-1-S-8, were isolated. S-1 was identified as 12β-hydroxypregna-4, 6, 16-triene-3, 20-dione (neridienone-A), obtained for the first time from Asclepiadaceous plants, by direct comparison with an authentic sample and the others were found to be steroidal glycosides. A new compound S-2A (named periplocogenin), and two known aglycones S-3A and S-5A, obtained by hydrolyzing the above glycosides with diluted acid, were respectively identified as 3β-O- (4', 6'-dideoxy-3'-O-methyl-Δ3'-D-2'-hexosulosyl) -Δ5-pregnene-17α, 20 (S) -diol, periplogenin, and Δ5-pregnene-3β, 20 (S) -diol from the proton and carbon-13 nuclear magnetic resonance spectral data. The structure of S-6A was identical with that of S-5A, and S-7A and S-8A were identical with S-2A.

Constituents of chinese crude drug "Wujiapi." VIII. On the structures of new oligosaccharides C1,D2,F1 and F2 of Bei-Wujiapi.

Four new type oligosaccharides C1, D2, F1 and F2 of Bei-Wujiapi (cortex of Periploca sepium BGE.) were elucidated to be 2-O-acetyl-β-D-digitalopyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-L-oleandronic acid-δ-lactone (Ia), 2-O-acetyl-β-D-digitalopyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-β-D-canaropyranosyl (1→4)-β-D-digitoxopyranosyl (1→4)-L-oleandronic acid-δ-lactone (IIa), β-D-digitalopyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-L-oleandronic acid-δ-lactone (IIIa) and β-D-digitalopyranosyl (1→4)-β-D-cymaropyranosyl (1→4)-β-D-canaropyranosyl (1→4)-β-D-digitoxopyranosyl (1→4)-L-oleandronic acid-δ-lactone (IVa). It should be noted that Ia, IIa, IIIa and IVa are the first oligosaccharides composed of 2, 6-dideoxysugars and 2, 6-dideoxyaldonic lactone. Furthermore, it is interesting that the sugar sequences of these oligosaccharides are ruled by the regularity in cardiac and pregnane type glycosides of Asclepiadaceous plants.

Constituents of Chinese Crude Drug “Wujiapi” . IV. On the Structure of a New Acetylbiose from Steroidal Glycosides of Bei-Wujiapi

The chemical structures of two hydrolysis products of the crude glycoside fractiion of Chinese crude drug “Bei-Wujiapi (cortex of Periploca sepium BGE.)” were studied. The one product is 4-O-(2-O-acetyl-β-D-digitalopyranosyl)-D-cymaropyranose and the other is methyl 4-O-(2-O-acetyl-β-D-digitalopyranosyl)-β-D-cymaropyranoside.

Constituents of Chinese crude drug "Wujiapi". 3. On the structure of glycoside G and K of Bei-Wujiapi.

The chemical structure of glycoside G (I), C36H56O13, mp 232-233°, [α]19D+30.2°(EtOH) and glycoside K (V), C40H66O16, mp 240-241°, [α]20D -27.58°(MeOH) were isolated from Bei-Wujiapi (cortex of Periploca sepium BGE.) and established to be periplocin and Δ5-pregnene-3β, 20α-diol(20)-β-D-glucopyranosyl (1glu→6glu)-β-D-glucopyranosyl (1glu→2dig)-β-D-digitalopyranoside. It should be noted that glycoside K is the first example of the pregnane type glycoside whose sugar moiety links to the hydroxyl group other than C3-hydroxyl group of the steroidal aglycone.