In Silico Pharmacokinetics Properties and In Vitro Bioactivities of Pregnane Derivatives and Other Compounds From Stems of Caralluma speciosa

Abstract

Objectives

Caralluma speciosa (N.E. Br.) N.E. Br. (Asclepiadaceae) and other succulent plants of the genus Caralluma have mainly been known for their appetite suppressing and weight loss properties, due to the presence of pregnane compounds. This study aimed to investigate chemical constituents and evaluate antimicrobial and antioxidant activities of stems of C speciosa.

Methods

The chloroform and methanol stems extracts were subjected to silica gel chromatographic separation. The structures of isolated compounds were established by FT-IR, 1D-NMR, and GC-MS instruments, and in comparison with reported data. The antimicrobial and antioxidant activities were performed using agar disc diffusion and DPPH assaying methods.

Results

The phytochemical investigation afforded 5 compounds, 3 pregnane derivatives (3-5), and 2 other compounds, heptacos-1-ene (1) and di-(2-ethylhexyl) phthalate (2). The chloroform: methanol (1:1) extract showed a good antifungal activity against Candida albicans up to 25 mg/mL dose (9.00 ± 1.00 mm to 17.17 ± 1.04 mm), equivalent to ketoconazole standard (17.67 ± 2.52 mm at 0.01 mg/disc). Compounds 3 and 5 were found violated 3 of the Lipinski's rule of 5. Compounds 2 and 4 exhibited a carcinogenicity property; while compounds 3, 4, and 5 were found as immunotoxic isolates.

Conclusion

All the extracts and isolated compounds 1-5 showed a negligible antibacterial and antioxidant activities compared to the antibiotic chloramphenicol (7.3 ± 0.4 to 21.15 ± 0.5 mm) and antioxidant ascorbic acid (IC₅₀ value of 5.62 µg/mL). Hence, further biological activities study (including the anticancer, anti-inflammatory, and antifungal activities) would be needed on all the extracts and isolated compounds to look for better efficacy and predict the structure–activity relationship of the compounds.

Keywords

asclepiadaceae succulent plants steroidal compounds antiobesity property drug likeness property bioactivity

Introduction

Caralluma speciosa (N.E. Br.) N.E. Br. (Asclepiadaceae) is a succulent perennial herb which grows in Acacia woodland and bush land. It is found in Ethiopia, Sudan, Somalia, Uganda, Kenya, and Tanzania.¹ The genus Caralluma is composed of nearly 260 species which have been reported to be used mainly as food sources. In India, for example, fruits and young shoots of C edulis are consumed as a vegetable.² The plant species belonging to this genus are mainly consumed for the purpose of antiobesity and appetite suppression. For instance, C adscendens fimbriata is used as an appetite suppressant in India.³ The appetite suppressing and weight loss properties of Caralluma species are believed due to the presence of their major phytoconstituents, pregnane glycosides (C₂₁ steroidal compounds). Pregnane glycosides, previously reported from the succulent African plant, Hoodia species, are confirmed for their appetite suppressing and antiobesity property.⁴ Caralluma species have also been reported for their ethnobotanical uses to cure human ailments such as malaria, inflammation, hyperglycemia, ulcers, and cancer. The fresh stems of C attenuate and Caralluma stalagmifera, for example, are used against diabetes and migraine.⁵ Caralluma tuberculata and Caralluma fimbriata are also used to cure rheumatism, diabetes, leprosy, paralysis, joint pains, fever, inflammation, and pyretic.² In Ethiopia, the stems and sap of C speciosa are used for the treatment of skin cyst and tumor, gangrene, poison, swollen body, wound, and itching skin.⁶

Many pregnane glycosides and extracts of various species of the genus Caralluma have also been reported for their biological activities. For instance, pregnane glycosides isolated from C penicillata, C tuberculata, and C russelliana showed antitrypanosomal and antiplasmodial potency.⁷ Six compounds isolated from C tuberculata were tested for their antimalarial and antitrypanosomal activities.⁸ The ethanolic extract of C tuberculata was also found as protector of gastric mucosa. In addition, C arabica was reported for its anti-inflammatory, antihyperglycemic, antinociceptive, gastric mucosa protective, and antiulcer properties.⁹ C umbellata was also evaluated for anti-inflammatory and antinociceptive activity. Caralluma fimbriata was another species studied for its antifungal and anthelmintic activities.^2,10 According to the biological activity study reported by Jitwasinkul and Charoensuksai,¹¹ the aqueous ethanolic extract of fresh C speciosa showed cytotoxic, antiproliferative, and pro-apoptotic activities against head-and-neck (HN22) and colon (HT29) cancer cell lines.

The chemical constituents reported from the genus Caralluma are mainly dominated by pregnane glycosides. Besides, megastigmane glycosides and few flavones have also been isolated from some species of the genus.¹² For instance, 4 acylated pregnane glycosides were identified from the aerial parts of C russelliana.¹³ Five steroidal glycosides (stalagmosides) and 2 steroidal glycosides (indicosides) were reported from whole parts of C stalagmifera and C indica, respectively.¹⁴ In addition, 5 steroidal glycosides and 27 pregnane glycosides were reported from the whole part of C dalzielii.¹⁵ Moreover, 18 fatty acids, 4 hydrocarbons and a β-sitosterol were reported from C edulis.¹⁶ Seventy-four volatile compounds and several fatty acids were also identified from stems and fruits of C europaea.¹⁷ However, to the best of our knowledge, no research works were reported on the chemical constituents, and antimicrobial and antioxidant activities of the stems of C speciosa. Hence, this research work aimed to investigate the chemical constituents and evaluate the antimicrobial and antioxidant activities of the stems of C speciosa. The isolated compounds, reported herein, were also subjected to computational study to predict their drug-likeness, ADME, and toxicity properties using the SwissADME and ProTox-II property explorer software.

Results and Discussion

Structural Identification of Isolated Compounds 1-5

Previously reported phytochemical studies on the genus Caralluma have revealed that members of this genus have been dominated by the C₂₁ steroidal-like compounds, known as pregnanes.⁴ Such classes of compounds exhibit in common fused tetracyclic skeletal structure with mainly 2 methyl groups at C-10 and C-13 and 2 extended carbon-chain at C-17 position. Pregnane compounds differ from each other by the presence or absence of different sugar units and other aglycone esterified groups (such as acetyl, benzoyl, and tigloyl) at different positions of the basic skeleton.¹⁸ In the present investigation, 3 pregnane derivatives (3, 4, and 5) and 2 nonpregnane compounds (1 and 2) (Figure 1) were isolated from chloroform and methanol extracts. This is the first report of their isolation from stems of C speciosa. The detailed structural interpretation of the compounds 1-5 is discussed as follows:

Figure 1.

Structures of compounds 1-5 isolated from stems of Caralluma speciosa. Evaluation of in vitro antimicrobial activity.

Compound 1: It was isolated as yellow amorphous (104 mg); R_f 0.75 (n-Hexane/EtOAc/AcOH; 1:1:0.1).

From the ¹H, ¹³C, and DEPT-135 NMR spectra (Supplemental Figures S1, S2, and S3), aliphatic proton and carbon signals (including methyl group) were observed at δ_H 0.88 to 2.04 and δ_C 14.6 to 34.3 ppm, respectively. Briefly, in the ¹H spectrum, a multiplet methine signal at δ_H 5.80 (1H, H-2) and a doublet of doublet methylene signal at δ_H 4.96 (2H, dd, H-1, J = 16.4, 11.1 Hz) were appeared to indicate the presence of an olefinic bond. The proton spectrum also displayed 3 aliphatic methylene multiplet signals, integrated for 2, 4, and 42 protons, respectively, at δ_H 2.06 (2H, H-3), 1.38 (4H, H-4/26), and 1.25 (42H, H-5 to H-25) and a triplet methyl signal at δ_H 0.88 (3H, H-27, J = 12.87 Hz). The existence of the olefinic bond was also evidenced by the appearance of methine (=CH-) and methylene (=CH₂) carbon signals at δ_C 139.8 (C-2) and 114.5 (C-1), respectively, in the ¹³C and DEPT-135 spectra. The ¹³C and DEPT-135 spectra also showed 6 aliphatic carbon signals, which attributed to 25 carbons, at δ_C 34.3 (C-3), 32.4 (C-25), 29.9 (C-5 and C-6), 30.2 (C-4 and C-7 to C-24), 23.2 (C-26), and 14.6 (C-27). The absence of any quaternary carbon signal in the DEPT-135 spectrum also confirmed that compound 1 was a member of a straight chain alkene. The NMR spectral data were compiled and presented in Supplemental Table S1. In the IR spectrum (Supplemental Figure S4), an intense peak was observed at 2918 to 2848 cm⁻¹ indicative of CH-stretching of alkyl including methyl groups. Additionally, a strong -C = C- stretching and = CH- bending bands were shown at 1462 and 717 cm⁻¹, respectively. The GC-MS spectrum (Supplemental Figures S5 and S6) showed a major chromatogram peak at the retention time of 40.73 min and a weak molecular ion peak at m/z of 380.4 in the positive ion mode which was compatible with the molecular weight of 378 [M + 2]⁺. Besides, a base peak at m/z of 57.1 was observed which represents the chemical composition of C₄H₉ (MW, 57). Moreover, 3 fragment ions were appeared at m/z of 71.1, 85.1, and 55.1, which belong to the elemental compositions C₅H₁₁ (MW, 70), C₆H₁₃ (MW, 85), and C₄H₇ (MW, 55), respectively. This led to the assignation of entire molecular formula of compound 1 as C₂₇H₅₄. Based on the above spectral information, the structure of compound 1 was deduced as heptacos-1-ene (Figure 1).

Compound 2: It was found as dark amorphous (186 mg); R_f 0.32 (n-Hexane/EtOAc/AcOH; 1:1:0.1).

In the ¹H NMR spectrum (Supplemental Figure S7), 2 doublet of doublet signals were observed at δ_H 7.53 (2H, dd, J = 4.45, 4.87 Hz) and 7.70 (2H, dd, J = 4.45, 4.87) due to the protons of symmetrical ortho disubstituted aromatic ester with an AA’BB’ spin system. The spectrum also showed an intense multiplet proton signal at the chemical shift range of δ_H 1.25 to 1.36, integrated for a total of 16 protons, indicated the presence of 8 methylene (-CH₂-) protons in at similar chemical environment. In addition, aliphatic proton signal was observed at δ_H 4.21 (4H, br t, J = 12.38 Hz) for 2 set of oxygenated methylenes neighboring with a multiplet signal at δ_H 2.33 (2H) of methine (-CH-) protons. The ¹H spectrum also showed a doublet of doublet signal at δ_H 0.89 (6H, dd, J = 9.83, 12.04) due to 2 symmetric methyl protons adjacent to 2 nonequivalent methylene protons; and a triplet signal at δ_H 0.78 (6H, t, J = 8.56) for 2 symmetric methyl protons next to 2 equivalent methylene protons, each.

In the ¹³C spectrum (Supplemental Figure S8), 3 aromatic carbon signals at δ_C 128.6, 130.7 and 132.2 along with an ester carbonyl carbon signal at δ_C 167.6 were showed which were due to the presence of 2 symmetrically esterified benzoic aromatic groups; and this was supported by the DEPT-135 spectrum (Supplemental Figure S9) in which 2 quaternary carbon signals (δ_C 132.2 and 167.5) and a signal at δ_C 67.5 (C-1/1’) of 2 symmetric oxygenated methylene carbons were revealed. The DEPT-135 spectrum also indicated the presence of an intense methine carbon signal at δ_C 38.5 (C-2/2’) together with 2 consecutive methyl carbon signals at δ_C 10.8 and 13.9 ascribed to 2 equivalent methyl groups, each. The obtained NMR spectral data is presented in Supplemental Table S2.

The IR spectrum (Supplemental Figure S10) showed an absorption peak of the ester carbonyl group (C = O) of the phthalate unit at 1734cm⁻¹ together with the C-O and aromatic C-H stretching at wavenumbers of 1274 and 731 cm⁻¹, respectively. It also displayed an intense peak at 2918 to 2855 cm⁻¹ which represents the C-H stretching of the long chain alkyl groups. From the GC/MS spectrum (Supplemental Figures S11 and S12), a molecular ion peak at m/z 390 in the EI-MS and major peak with retention time of 37.031 min in the GC were appeared which is compatible with the molecular formula of C₂₄H₃₈O₄ (calculated MW, 390). A base peak at m/z 149.0 (calcd. 149.0 for C₈H₅O₃) was also obtained, including 4 relatively intense product ions at m/z 167.0 (C₈H₇O₄, calcd. MW, 167), 57.1 (C₄H₉, calcd. MW, 57), 71.0 (C₅H₁₁, calcd. MW, 71), and 279.0 (C₁₆H₂₃O₄, calcd. MW, 279). Based on the above spectral information and reported data,¹⁶ the structure of compound 2 was elucidated as di-(2-ethylhexyl) phthalate (Figure 1). This compound 2 is reported herein for the first time from the genus, Caralluma.

Compound 3: It was yielded as yellow-brown amorphous (104 mg); R_f 0.55 (DCM/EtOAc/AcOH; 3:1:0.1).

It is observed that the ¹H and ¹³C NMR spectra (Supplemental Figures S13 and S14) contained many complicated aliphatic proton and carbon signals at δ_H 0.88 to 2.02 and δ_C 10.5 to 55.7 ppm, respectively. This showed that compound 3 contained a pregnane nucleus in its entire structure. The ¹H spectrum also showed 3 multiplet signals at δ_H 3.33 (1H, H-3), 4.79 (1H, H-12), and 5.39 (1H, H-20) due to 3 oxymethinic protons at C-3, C-12, and C-20, respectively, of the pregnane skeleton. A hydroxylated quaternary carbon (-C-OH) signal at δ_C 86.5 was also observed in the DEPT-135 spectrum (Supplemental Figure S15), assigned to C-14 of the pregnane skeleton.¹⁸ Besides, the 3 neighboring singlet proton signals observed at δ_H 1.04 (3H, H-18), 0.88 (3H, H-19), and 1.24 (3H, H-21) assigned to 3 methyl groups at C-18, C-19, and C-21, respectively, of the pregnane backbone; and a multiplet signal shown at the δ_H range of 1.64 to 1.79 belonged to 3 methine protons (H-5, H-8, and H-9) of C-5, C-8, and C-9, and a methylene protons (H-11) at C-11 (Supplemental Table S3). The obtained overall spectral data of the pregnane part are in close agreement with information in literature.¹⁸ The ¹H spectrum also showed the presence of 2 consecutive doublet of doublet and broad triplet signals at δ_H 7.92 (1H, dd, H-3’/7’; J = 7.74, 7.74 Hz) and 8.18 (1H, br t, H-3’/7’; J = 18.32 Hz) integrated for 2 protons each; 2 pseudo triplet signals at δ_H 7.28 (1H, t, H-4’/6’; J = 12.95 Hz) and 7.34 (1H, t, H-4’/6’; J = 12.95 Hz) integrated for 2 protons each; and triplet of triplet and broad quartet signals at δ_H 7.59 (1H, tt, H-5’; J = 12.95, 14.10 Hz) and 7.73 (1H, br q, H-5’; J = 26.07 Hz) which were due to ortho, metha, and para protons (at C-3’/7’, C-4’/6’, and C-5’, respectively) of 2 benzoyl units with AA’BB’C type of spin system. This confirmed the pregnane core possessed 2 set of esterified groups positioned at C-12 and C-20 (Supplemental Table S3).

From the ¹³C spectrum, those 3 signals shown at δ_C 73.8, 86.5, and 70.4 were attributed to 3 oxygenated carbon signals (C-12, C-14, and C-20, respectively) of the pregnane part. Besides, the 3 consecutive signals observed at δ_C 10.5, 12.5, and 19.2 were because of the presence of 3 methyl carbons (C-18, C-19, and C-21, respectively) of the pregnane unit. Furthermore, 2 close signals at δ_C 129.2 and 129.6, 127.9 and 128.3, and 132.3 and 132.8 were the ortho (C-3’/7’), metha (C-4’/6’), and para (C-5’) carbons of the 2 benzoyl groups attached at C-12 and C-20, respectively. The presence of the 2 ester groups of the benzoyl moieties (at C-12 and C-20) was also witnessed by the presence of 2 ester carbonyl carbon signals at δ_C 165.8 (C-12) and 166.4 (C-20) in the ¹³C spectrum. In the DEPT-135 spectrum, 7 quaternary signals were confirmed; 3 signals at δ_C 35.2, 53.2, and 86.5 due to the C-10, C-13, and C-14 (oxygenated with OH) of the pregnane unit; 2 signals at δ_C 130.4 and 130.5 belong to C-2’ of the 2 benzoyl groups; and the remaining 2 signals at δ_C 165.8 and 166.4 due to the 2 carbonyl carbons of the benzoyl ester groups (Supplemental Table S3). The overall ¹³C and DEPT-135 data of the aglycone part of compound 3 were found to be in a good correlation with the one from literature.¹⁸

The ¹H spectrum also showed many multiplet signals of oxymethinic protons in the chemical shift range of 3.53 to 4.98 ppm. This indicated the presence of sugar moieties within the pregnane unit. The number of consecutively connected saccharide units can be recognized by the number of anomeric carbon signals at around δ_C 90 to 110 and anomeric proton signals at δ_H 4 to 5 ppm.¹⁸ In our case, the presence of 2 intense carbon signals at δ_C 95.3 (C-1’’) and 99.1(C-1’’’) in the ¹³C spectrum together with 2 proton signals at δ_H 4.98 (1H, br d, H-1’’, J = 10.52 Hz) and 4.90 (1H, br d, H-1’’’, J = 6.89 Hz) in the ¹H spectrum inferred that there were 2 anomeric carbons and associated protons which belong to 2 interlinked sugar moieties. In addition, 2 proximate carbon signals at δ_C 57.0 (C-7’’) and 57.7 (C-7’’’) along with 2 intense singlet proton signals at δ_H 3.53 (3H, s, H-7’’) and 3.54 (3H, s, H-7’’’) were observed which ascribed to 2 methoxy (MeO-) groups. This revealed that the 2 sugar units were cymarose-I (C-1’’) and cymarose-II (C-1’’’).¹⁸ Besides, the presence of 2 methyl groups at δ_C 17.9 and 18.1 (C-6’’ and C-6’’’, respectively) and at δ_H 1.32 (6H, br s, H-6’’ and H-6’’’) in combination with the methylene carbon signal (δ_C 36.1, C-5’’ and C-5’’’) and multiplet proton signal (δ_H 1.79, 4H, m, H-5’’ and H-5’’’) was additional evidence to the 2 sugar moieties to be cymarose-I and II.¹⁸ In reference to reported data for similar compound,¹⁸ the disaccharide units were attached to the pregnane unit at C-3 (δ_C 72.2). The IR spectrum (Supplemental Figure S16) also provided supportive information by showing major bands at 3420, 2918 to 2848, 1713, and 710 cm⁻¹ of the hydroxyl, alkyl, carbonyl, and aromatic groups, respectively. Based on the spectroscopic data (Supplemental Tables S3 and S4) and comparison with reported data,^18–20 the structure of compound 3 was proposed as 12β,20-O-dibenzoly-14β-hydroxy-5α,6dihydropregane-3-O-β-cymaropyranosyl-(1-4)-β-cymaropyranoside (Figure 1).

Compound 4: It was isolated as yellowish amorphous (127 mg); R_f 0.35 (n-Hexane/EtOAc/AcOH; 1:1:0.1).

The acquired ¹H, ¹³C, and DEPT-135 spectra (Supplemental Figures S17, S18, and S19) confirmed the presence of a pregnane aglycone nucleus as in compound 3. Unlike compounds 3 and 5, however, no glycoside units were observed and instead an olefinic bond between C-20 and C-21 was presented in the pregnane skeleton of compound 4. This was evidenced by the appearance of a multiplet at δ_H 6.83 (1H, H-20) and 2 doublet at δ_H 5.33 (1H, d, H-21, J = 15.20 Hz, cis) and 5.78 (1H, d, H-21, J = 17.40 Hz, trans) proton signals in the ¹H spectrum which belong to the olefinic protons (methine and methylene) of the pregnane part. The ¹³C spectrum also showed 2 carbon signals at δ_C 139.3 (C-20) and 114.1 (C-21) for terminal olefinic methine (=CH-) and methylene (=CH₂) carbons, respectively, (Supplemental Table S5). Furthermore, the NMR spectra also showed methylenedioxy-benzyl ester moiety positioned at C-12 of the aglycone pregnane unit of compound 4. This was confirmed by the presence of an oxygenated methylene carbon signal at δ_C 68.3 (C-22) in the ¹³C and DEPT-135 spectra, and methylene proton signal at δ_H 4.98 (2H) in the ¹H spectrum. Moreover, a carbon signal at δ_C 167.9 (C-1’) from the ¹³C spectrum, for the ester carbonyl, a broad singlet at δ_H 8.04 (2H, H-3’/7’), and 2 broad triplets at δ_H 7.57 (2H, br t, H-4’/6’, J = 17.70 Hz) and 7.80 (1H, br t, H-5’, J = 16.82 Hz) proton signals in the ¹H spectrum were observed to indicate the presence of an esterified benzoyl substituent.

But, herein, it is very important to note that the attachment of the benzoyl ester moiety outside the pregnane aglycone nucleus (C-22) of compound 4 was unprecedented in other pregnane compounds. That is, pregnane compounds are known by containing glycoside and/or nonsugar (such as benzoyl, acetoyl, and tigloyl) moieties attached either within the tetracyclic rings or at C-20 of the pregnane skeleton. The phenomenon in which the benzoyl ester moiety presented at C-22 of compound 4 might be either due to the presence of additional methyl group at C-12 or a methyl ester of benzoyl substituent which latter was incorporated into the nonmethylated (at C-12) of the basic pregnane structure. The compiled ¹H, ¹³C, and DEPT-135 spectral data of compound 4 are presented in Supplemental Table S5. The IR absorption bands at 1733, 1274, and 710 cm⁻¹ (Supplemental Figure S20) were suggestive of ester carbonyl, C-O, and aromatic group of the esterified phenyl moiety. The absorption peaks shown at 2918 to 2848 and 1462 cm⁻¹ were belong to the stretching of C-H alky groups and C = C, respectively, of the pregnane skeleton. The GC analysis (Supplemental Figure S21) showed a major peak at retention time of 45.01 min with a corresponding weak molecular ion peak at m/z 426 [M + 6]⁺ of the ES-MS (Supplemental Figure S22). This is matched with the proposed molecular formula (C₂₉H₄₀O₂, calculated MW, 420). Besides, a base peak at m/z 97.1 (C₇H₁₃, MW: 97) is observed together with 3 intense fragment ions at m/z of 57.1 (C₄H₉, MW: 57), 83.1 (C₆H₁₁, calculated MW 83), and 69.1 (C₅H₉, MW: 69). On the basis of the above information, we proposed the chemical structure of compound 4 as hexadecahydro-10,13-dimethyl-17-vinyl-1H cyclopenta[a]phenanthren-12-yl)methyl benzoate (Figure 1).

Compound 5: It was obtained as brown amorphous (157 mg); R_f 0.42 (DCM/EtOAc/AcOH; 9:1:0.1).

The ¹H, ¹³C, and DEPT-135 spectral feature (Supplemental Figures S23, S24, and S25) of compound 5 (Supplemental Tables S6 and S7) was found to be resemble to that of compound 3. That is, in compound 5 also, 2 set of benzoyl ester groups attached to the pregnane skeleton at C-12 and C-20 were observed in which their spectral data were compatible with that of compound 3 with a slight change both in chemical shift and multiplicities. The only notable difference was the presence of 2 exclusive spectral information observed from the spectral result of compound 5. The first one was the presence of an additional terminal sugar unit assigned as thevetose (thev) identified by the presence of 3 additional major carbon signals at δ_C 104.7, 61.0, and 18.2 attributed to another anomeric (C-1’’’’’), methoxy (C-7’’’’’), and methyl (C-6’’’’’) carbons (Supplemental Table S7). The presence of this glycoside unit was further confirmed by the corresponding broad doublet (δ_H 4.27, 1H, br d, H-1’’’’’, J = 4.26 Hz), singlet (δ_H 3.77, 3H, s, H-7’’’’’), and broad singlet (δ_H 1.34, 3H, br s, H-6’’’’’) signals of the anomeric, methoxy, and methyl protons, respectively, in the ¹H spectrum. The second information was the incorporation of an acetyl moiety to the pregnane aglycone skeleton of compound 5 attached at C-14 (δ_C 87.1). This was witnessed by the presence of a deshielded signal of methyl protons at δ_H 2.81 (3H, s, H-2’’) along with their carbon signal at δ_C 22.3 (C-2’’) in the ¹H and ¹³C NMR spectra (Supplemental Table S6). Moreover, a downshielded carbon signal at δ_C 175.7 (C-1’’) was recognized in the ¹³C and DEPT-135 spectra due to the quaternary carbonyl carbon of the acetyl group.¹⁸ The small increment in chemical shift (+0.6 ppm) of C-14 (δ_C 87.1) in this compound in comparison to C-14 (δ_C 86.5) of compound 3 might also be further assertion to the incorporation of acetyl moiety to the pregnane nucleus of compound 5 at C-14.¹⁸ Some supportive absorption bands were also observed in the IR spectrum (Supplemental Figure S26) at wavenumbers (cm⁻¹) of 3426 (OH stretches), 2918-2848 (alkyl C-H stretching), 1713 (carbonyl groups), 1455 (C-O stretching), and 710 (aromatic groups). The compiled spectral data of compound 5 are shown in Supplemental Tables S6 and S7. Based on the spectral data presented above, the structure of compound 5 was identified to be 12β,20-O-dibenzoly-14β-acetyl-5α,6-dihydropregane-3-O-β-thevetopyranosyl-(1-4)-β-cymaropyranosyl-(1-4)-β-cymaropyranoside (Figure 1).

Bacteriological Assay

The antibacterial activity results of 5 crude extracts (n-hexane, chloroform, 1:1 of chloroform: methanol, methanol, and ethanol) and 5 isolated compounds 1-5 (Figure 2) of the stems of C speciosa are presented in Supplemental Tables S8 and S9, respectively.

Figure 2.

DPPH radical scavenging percentage (%) versus different concentrations of Caralluma speciosa stems extracts and ascorbic acid. HE, n-hexane extract; CE, chloroform extract; CME, 1:1 of chloroform: methanol extract; ME, methanol extract; EE, ethanol extract; AA, ascorbic acid.

As can be indicated in Supplemental Table S8, the best diameter of inhibition zone (8.65 ± 1.63 mm) against Escherichia coli was recorded by chloroform and then followed by methanol (7.09 ± 0.88 mm) extracts at the maximum concentration (100 mg/mL). Whereas the n-hexane, chloroform: methanol (1:1), and ethanol extracts (EEs) did not show any activity against E coli at all tested concentrations. However, the mean difference between the extracts was not significant (P > .05). All tested extracts were found with no activity against the methicillin-resistant Staphylococcus aureus bacterium at all concentrations, except the EE which tried to show a significantly different (P < .05) slight inhibitory effect (7.55 ± 0.25 mm) at the higher concentration (100 mg/mL). On the other hand, the Gram-negative Pseudomonas aeruginosa bacterium showed a total resistant against all evaluated extracts at all concentrations.

All the isolated compounds 1-5 were found with no activity against the Gram-negative bacteria, E coli and P aeruginosa, at doses up to 1 mg/mL (Supplemental Table S9). Whereas all these compounds exhibited a moderate activity (diameter of inhibition zone >7 mm) against the inflammation causing Gram-positive S aureus bacterium, with higher inhibitory value (11.03 ± 0.6) showed by compound 5, then by compound 3 (9.20 ± 0.76) followed by compound 2 (9.08 ± 0.06) at the maximum concentration (1 mg/mL). The observed susceptibly difference between the Gram-positive and Gram-negative bacteria toward the tested compounds can be related to the variation in biological makeup of the bacteria's outer cell membrane layer. That is, the outer cell membrane of Gram-positive bacteria constitutes simple and thin layers of peptidoglycans which can easily be penetrated by chemical agents. Whereas outer cell membrane of Gram-negative bacteria is built from multilayered peptidoglycan and phospholipids which is impermeable to especially less effective compounds.²¹ Besides, glycosylated and hydroxylated pregnane compounds may exhibit better activity against Gram-positive bacteria due to the hydroxyl groups which can form a hydrogen bonding with phospholipidic membrane of the bacteria leading to the integration and complex formation of the compounds. This fact may hold true for compounds 3 and 5, herein, which showed better activity against S aureus.

Antifungal Activity Assay

The chloroform: methanol (1:1) extract exhibited prominent inhibitory potential against Candida albicans at all evaluated concentrations; except for the minimum concentration (12.5 mg/mL) which showed zero diameter of inhibition zone. This extract recorded the inhibitory values of 9.00 ± 1.00 mm for 25 mg/mL, 14.67 ± 0.58 mm for 50 mg/mL, and 17.17 ± 1.04 mm for 100 mg/mL. The mentioned extract in general was found as active as the standard antifungal drug ketoconazole 0.01 mg/disc (17.67 ± 2.52 mm) mainly at the maximum concentration.

Examination of Antioxidant Activity

The crude extracts and isolated compounds 1-5 were assessed for their antioxidant effectiveness using the DPPH free radical and potassium ferric reducing antioxidant power (PFRAP) assaying techniques. Ascorbic acid (AA) and sample-free DPPH solution in methanol were used as positive and negative controls, respectively.

DPPH Free Radical Scavenging Activity Assay

The obtained results (Supplemental Tables S10 and S11, and Figures 2 and 3) revealed that each tested analyte showed a directly correlated (dose-dependent) activity, that is, the DPPH radical scavenging activity percentage was getting increased as the concentration increased. Ethanol extract showed a better DPPH radical scavenging percentage (58.092 ± 0.00) followed by chloroform extract (CE, 51.303 ± 0.04) in comparison to AA (95.342 ± 0.02) at the highest concentration of 500 µg/mL (Supplemental Table S10 and Figure 2). The inhibitory activity of the tested extracts against the DPPH radicals was generally observed negligible with higher IC₅₀ values in reference to the standard AA (5.62 µg/mL).

Figure 3.

DPPH radical scavenging percentage (%) versus different concentrations (µg/mL) of isolated compounds 1-5 and ascorbic acid (AA).

Among the isolated compounds, compound 4 recorded better DPPH radical scavenging percentage (86.225 ± 0.09), followed by compound 3 (72.597 ± 0.11) and compound 5 (56.13 ± 0.11) at the highest concentration (500 µg/mL) with IC₅₀ values of 15.67, 73.48, and 270.02 µg/mL, respectively (Supplemental Table S11 and Figure 3). The DPPH radical scavenging potency of the isolated compounds 1-5 was generally found to be very low compared to AA (IC₅₀ value of 5.62 µg/mL).

Potassium ferric reducing antioxidant power assay

The obtained PFRAP results of tested extracts and isolated compounds 1-5 are presented in Supplemental Tables S12 and S13, respectively. As shown from the tables, the ferric reducing power (expressed in absorbance at 700 nm) of each tested compound and extract showed a positive correlation with corresponding concentrations (from 25 to 500 µg/mL). That is, at high concentration, an intense green color with greater absorbance of mixtures was observed indicates higher ferric ion reducing potential. Among the extracts, EE showed a slightly higher reducing activity (0.497 ± 0.001) at a higher concentration of 100 µg/mL. Chloroform extract exhibited the next higher reducing power (0.488 ± 0.001) at similar concentration. However, with respect to AA, recorded PFRAP of all the extracts was found to be very weak at each corresponding concentration. Out of the compounds, compound 2 recorded highest PFRAP with an absorbance of 1.622 ± 0.004 followed by compound 3 (1.608 ± 0.002) at the highest concentration (500 µg/mL), whereas the lowest PFRAP (0.291 ± 0.001) was observed in compound 1 at same concentration (Supplemental Table S13). However, in comparison to AA at each corresponding concentration, the obtained result showed a weak ferric reducing antioxidant power of each compound.

Drug-Likeness, ADME, and Toxicity Properties of Isolated Compounds 1-5

The obtained in silico drug-likeness, ADME, and toxicity properties of the isolated compounds 1-5 are presented in Supplemental Tables S14, S15, and S16, respectively. The drug-likeness property result (Supplemental Table S14) revealed that compounds 3 and 5 violated, each, 3 of the Lipinski's rule of 5 (with LogP > 4.45, number of hydrogen acceptor > 10, and molecular weight > 500 g/mol). Whereas compounds 1, 2, and 4 disobeyed one rule (LogP > 4.45). The pharmacokinetics property (Supplemental Table S15) indicated that all, except compound 2, were found with low gastrointestinal absorption; and all of them could not cross the blood–brain barrier. Compounds 1, 2, and 5 were found as p-glycoprotein substrates, and all of the compounds were observed as noninhibitors of the CYP1A2, CYP2C19, and CYP2D6 cytochrome enzymes. But, compound 2 was found as inhibitor of CYP2C9 and CYP3A4. Besides, compounds 3 and 5 were found as CYP3A4 inhibitors and compound 4 showed an inhibitory effect against CYP2C9. According to the toxicity property prediction (Supplemental Table S16), compound 4 was found within the toxicity class of 4 (LD₅₀ value of 1340 mg/kg). Compounds 2 and 4 exhibited a carcinogenicity property; while compounds 3, 4, and 5 were found as immunotoxic agents.

In this study, both the in vitro and in vivo anticancer activity of the studied C speciosa stems were not included to support the claimed traditional medicinal use against tumor. Besides, the experimental cytotoxicity effect of the isolated compounds was not studied herein based on the obtained computational study result. Furthermore, the plant extracts and isolated compounds were evaluated against few microbial strains and oxidants. All these points could be considered as the limitations of the present study.

Conclusions

The phytochemical investigation resulted in isolation of 3 pregnane derivatives (3-5) and 2 nonpregnane compounds (1 and 2). All the extracts and isolated compounds 1-5 showed a negligible antibacterial and antioxidant activity compared to the antibiotic chloramphenicol (7.3 ± 0.4 to 21.15 ± 0.5 mm) and antioxidant AA (IC₅₀ value of 5.62 µg/mL). Whereas the chloroform: methanol (1:1) extract showed a good antifungal activity against C albicans up to 25 mg/mL, comparable to the standard Ketoconazole (17.67 ± 2.52 mm at 0.01 mg/disc). The computational study revealed that compounds 3 and 5 violated 3 of the Lipinski's rule of 5. Compounds 2 and 4 exhibited a carcinogenicity property; while compounds 3, 4, and 5 were found as immunotoxic isolates. It is recommendable to conduct further biological activities, including the anticancer, anti-inflammatory, and antifungal activities, of all the extracts and isolated compounds to discover additional bioactive chemical constituents and predict their structure–activity relationship.

Experimental

Plant Material

The stems of C speciosa (Figure 4) were collected during October 2022 from the prehistoric Harla town and its surroundings, Eastern Ethiopia, located at a distance of 515 km from the capital Addis Ababa. The area is found with the geographical coordinates of 9°27′ and 9°39′N latitude, and 41°38′ and 42°20′E longitude at elevation of 950 to 2260 m above sea level. The mean annual temperature and rainfall are about 22.8 °C and 1000 mm, respectively.⁶ The taxonomy of the plant was identified and a voucher specimen (voucher number, AHU111) was preserved in the herbarium of Haramaya University. Collected stems (2 kg) were washed with tap water, dried in shade at room temperature for 2 weeks, and ground into powder with electrical blender. Powdered samples were stored in a glass container and kept at 4 °C refrigerator for further experimental activities.

Figure 4.

Picture of Caralluma speciosa (photo by Tsegu K., October, 2022).

Chemicals and Instruments

Various chemicals such as the TLC detecting agents (iodine vapor, vanillin/MeOH/H₂SO₄), analytical grade organic solvents (methanol, ethanol, n-hexane, ethyl acetate, chloroform, and dichloromethane), inorganic reagents (ferric chloride, potassium ferricyanide, and 0.2 M potassium phosphate buffer), culture media (MHA and PDA), and others (trichloroacetic acid, DMSO, chloramphenicol standard, ketoconazole standard, 230-400 mesh size normal silica gel, AA, acetic acid, and DPPH free radical) were used. Silica gel 60 F₂₅₄ (Merck) precoated aluminum TLC sheet, glass column, electrical laboratory blender, suction filtration apparatus, Petri plates, orbital shaker (Hy-5A, Movel Scientific Instrument Co. Ltd), rotary evaporator (Rotary vacuum, Jainsons), incubator (Binder B28), autoclave (tuttnauer 3150EL), UV-lamp cabinet (UVP Chromato-Vue C-70G, Analytik Jena), UV-Vis spectrophotometer (Cecil CE4001 UV/VIS), Spectrum 65 FT-IR (PerkinElmer, 4000-400 cm⁻¹), GC-MS spectrometry (7890B and 5977A, Agilent Technologies), and ¹H (400 MHz) and ¹³C (100 MHz) NMR spectroscopy were employed.

Extraction and Chromatographic Separation

Coarse powder of C speciosa stems (500 g) was extracted with n-hexane (3×, 2.5 L) via shaking on orbital shaker for 24 h. The extract solution was then filtered by suction filtration, and solvent was evaporated by rotary evaporator resulted in yellow n-hexane extract (6 g). The remained n-hexane residue was consecutively extracted with chloroform, chloroform: methanol (1:1), ethanol, and methanol using the same extraction steps which afforded the corresponding yield of 19, 18.9, 8, and 10 g. The TLC profiles of all the extracts were examined using the solvent systems: n-hexane/DCM/AcOH (1:1:0.1), DCM/EtOAc/AcOH (1:1:0.1), and EtOAc/MeOH/AcOH (3:1:0.1); and the visualizing techniques: UV-lamp (254 and 365 nm) followed by iodine vapor. Thereafter, the chloroform and methanol extracts were fractionated over column chromatography (CC) normal silica gel as follows.

The chloroform extract (15 g) was reconstituted in chloroform (100 mL), adsorbed on silica gel (20 g), and dried using rotary evaporator. Adsorbed and concentrated sample was then applied onto CC silica gel (200 g). Three hundred twenty-five fractions were collected using the eluents n-hexane/DCM/EtOAc/acetone with gradient of polarity. Among the fractions, Fr.1-25 (eluted with n-hexane), Fr.88-118 (eluted with DCM), and Fr.204-211 (eluted with DCM/EtOAc, 1:1) yielded compounds 1 (104 mg), 2 (186 mg), and 3 (104 mg), respectively. Fr.119-203 (yellowish amorphous, 600 mg) was further rechromatographed using the same CC silica gel technique. Out of the collected 135 subfractions, sub-fr70-120 (eluted with CHCl₃/EtOAc, 3:2) yielded compound 4 (127 mg).

The methanol extract (9 g) was dissolved in methanol (25 mL), adsorbed on silica gel (10 g), and fractionated over CC silica gel (150 g) using DCM/EtOAc/MeOH as eluents in increasing ratio of polarity. As a result, 167 fractions were collected, out of which Fr.74-83 (eluted with DCM/EtOAc, 1:4) furnished compound 5 (157 mg). The structures of the isolated compounds 1-5 were elucidated by FT-IR, GC-MS, and 1D-NMR instrumental analyses along with reported data in literature. All the instrumental analysis settings were similar to the previous one described by Kiros et al.²²

Evaluation of In Vitro Antimicrobial Activity

Bacteriological assay

The antibacterial activity of all extracts and isolated compounds 1-5 was evaluated against S aureus ATCC 25923, E coli ATCC 25922, and P aeruginosa ATCC 27853 standard human bacterial pathogens obtained from Ethiopian public health institute. Experimental activity was conducted at Microbiology Laboratory, School of Medical Laboratory, Haramaya University. Mueller Hinton Agar (MHA) disc-diffusion technique was followed according to the standard protocols of Clinical and Laboratory Standards Institute.²³ Three concentrations (50, 25, and 12.5 mg/mL) of each extract were prepared from corresponding stock solutions (200 mg in 2 mL 4% DMSO) using the 2-fold serial dilution method.^22,24 Similarly, 4 concentrations (0.5, 0.3, 0.1, and 0.05 mg/mL) of each isolated compound were prepared from respective stock solutions (5 mg in 5 mL 4% DMSO). The experimental procedures stated in Kiros et al²² were also used in this study for the remaining detailed activities.

Antifungal activity assay

The chloroform: methanol (1:1) extract was assessed for its potential antifungal activity against C albicans ATCC10231 using the disc diffusion method mentioned above. A PDA medium was prepared as per the instruction of the manufacturer. The PDA-containing plate was then inoculated with C albicans suspension by streaking with a sterilized swab. Similar to the antibacterial activity experiment, 4 concentrations (12.5, 25, 50, and 100 mg/mL) were prepared in DMSO and 100 µL amount of each concentration was loaded onto sterile Whatman No. 1 filter paper disc (6 mm). Thereafter, the impregnated discs were placed onto the surface of the inoculated agar plates using sterile forceps. Commercial Ketoconazole/Tilt disc (0.01 mg/disc) was used as a standard drug. Then the PDA plates were sealed with Para film and incubated at 27 °C for 3 to 5 days. After incubation, the diameters of the zone of inhibition around each disc were measured using caliper (in mm). Experiments were conducted in duplicate, and results were expressed in mean and standard deviation.

Examination of antioxidant activity

The antioxidative effect of the extracts and isolated compounds 1-5 was also evaluated using DPPH free radical and PFRAP assays.

DPPH free radical scavenging activity assay

The DPPH free radical trapping power of the extracts and isolated compounds 1-5 was evaluated against 6 concentrations (500, 250, 150, 100, 50, and 25 µg/mL in methanol) using the procedures described previuosly^22,25 and compared with AA.

Potassium ferric reducing antioxidant power assay

In the present study, ferric ion reducing power of the extracts and isolated compounds 1-5 was also studied at 6 different concentrations (500, 250, 150, 100, 50, and 25 µg/mL in H₂O) by employing the experimental procedure stated earlier.^22,26

General information of the isolated compounds 1-5

Heptacos-1-ene (1): Yellow amorphous (104 mg); R_f 0.75 (n-Hexane/EtOAc/AcOH; 1:1:0.1); IR ν˜ [cm⁻¹]: 2918-2848 (CH- stretching), 1462 (C = C stretching), 912-717 (=CH- bending); See Supplemental Table S1 for detailed ¹H, ¹³C, and DEPT-135 spectral data; GC-MS spectrum showed a major peak at retention time of 40.73 min with EI-MS m/z (relative intensity in %): weak [M + 2]⁺ 380.4 (C₂₇H₅₄, MW: 378), 57.1 (100), 70.1 (79.28), 85.1 (61.46), and 55.1 (22.02).

Di-(2-ethylhexyl) phthalate (2): Dark amorphous (186 mg); R_f 0.32 (n-Hexane/EtOAc/AcOH; 1:1:0.1); IR ν˜ [cm⁻¹]: 2918-2855 (CH- stretching), 1734 (C = O ester), 1274 (ester C-O stretching), 731 (aromatic group); Detailed ¹H, ¹³C, and DEPT-135 spectral data are shown in Supplemental Table S2; EI-MS m/z (relative intensity in %): weak [M]⁺ 390 (C₂₄H₃₈O₄, MW: 390) with the GC retention time of 37.85 min, 149.0 (100), 57.1 (48.69), 71.0 (34.42), and 279.0 (27.05).

12β,20-O-dibenzoly-14β-hydroxy-5α, 6 dihydropregane-3-O-β-cymaropyranosyl-(1-4)-β-cymaropyranoside (3): Yellow brown amorphous (104 mg); R_f 0.55 (DCM/EtOAc/AcOH; 3:1:0.1); IR ν˜ [cm⁻¹]: 3420 (OH), 2918-2848 (alkyl CH stretching), 1713 (ester C = O), 1448 (C-O), 710 (aromatic group); See Supplemental Tables S3 and S4 for detailed ¹H, ¹³C, and DEPT-135 spectral data.

Hexadecahydro-10, 13-dimethyl-17-vinyl-1H cyclopenta[a]phenanthren-12-yl) methyl benzoate (4): Yellowish amorphous (127 mg); R_f 0.35 (n-Hexane/EtOAc/AcOH; 1:1:0.1). IR ν˜ [cm⁻¹]: 2918-2848 (CH stretching), 1733 (C = O ester), 1462-1274 (ester C-O stretching), and 710 (aromatic bending); See Supplemental Table S5 for detailed ¹H, ¹³C, and DEPT-135 spectral data; GC-MS spectrum at retention time of 45.01 min and EI-MS m/z (intensity in %): 426 [molecular ion peak + 6]⁺ which is matched with the molecular formula C₂₉H₄₀O₂ (calculated MW, 420), 97.1 (100), 57.1 (98.12), 83.1 (82.51), and 69.1 (64.41).

12β,20-O-dibenzoly-14β-acetyl-5α,6-dihydropregane-3-O-β-thevetopyranosyl-(1-4)-β-cymaropyranosyl-(1-4)-β-cymaropyranoside (5): Brown amorphous (157 mg); R_f 0.42 (DCM/EtOAc/AcOH; 9:1:0.1); IR ν˜ [cm⁻¹]: 3426 (OH groups), 2918-2848 (alkyl groups), 1713 (C = O ester), and 710 (aromatic groups); ¹H, ¹³C, and DEPT-135 spectral data: See Supplemental Tables S6 and S7.

Statistical Data Analysis

The mean and standard deviation of antimicrobial and antioxidant activities were performed by descriptive statistics of SPSS version 20 software. The mean difference between the extracts was analyzed by one-way analysis of variance (ANOVA) using the Tukey HSD test and was considered as significant at 0.05 level.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231220110 - Supplemental material for In Silico Pharmacokinetics Properties and In Vitro Bioactivities of Pregnane Derivatives and Other Compounds From Stems of Caralluma speciosa

Supplemental material, sj-docx-1-npx-10.1177_1934578X231220110 for In Silico Pharmacokinetics Properties and In Vitro Bioactivities of Pregnane Derivatives and Other Compounds From Stems of Caralluma speciosa by Tsegu Kiros, Seid Mohammed, Aman Dekebo and Yadessa Melaku in Natural Product Communications

Footnotes

Acknowledgments

The authors are thankful to Dr Mesfin Getachew, Addis Ababa Science and Technology University, and Dr Yonas Chebude, Addis Ababa University, Ethiopia, for conducting the NMR and FT-IR spectral analysis, respectively. The authors are also grateful to Dr Anteneh Belayneh, Haramaya University, Ethiopia, for the botanical identification of studied plant.

Authors’ Note

1D-NMR, IR, and GC-MS spectra and in silico pharmacokinetics properties of compounds 1-5, and in vitro antibacterial and antioxidant activities data of these compounds and extracts are available online.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Adama Science and Technology University under Grant (grant number ASTU/AS-R/003/2020); and the World Academy of Sciences (TWAS) and the United Nations Educational, Scientific and Cultural Organization (UNESCO) under the TWAS Research Grant (grant number RGA No. 20-274 RG/CHE/AF/AC_G – FR3240314163).

ORCID iD

Aman Dekebo

Supplemental Material

Supplemental material for this article is available online.

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