Isolation and characterization of bioactive components from Mirabilis jalapa L. radix.

The present investigation was carried out to isolate and characterize bioactive components from Mirabilis jalapa L. radix ( zǐ mò lì gēn). Thin-layer chromatography was used for the separation of spots from fractions of the crude extract. Separated spots were collected for identification of their activities. Free-radical scavenging activity was evaluated by spraying thin-layer chromatography plates (spotted with fractions) with 0.2% of 2,2-diphenyl-1-picrylhydrazyl solution. Activity against human pathogens such as Staphylococcus aureus and Candida albicans were determined using the agar diffusion method. Potential spots were subjected to infrared (IR) analysis and gas chromatography for characterization. Two spots (5F1 and 1F3) showed free-radical scavenging activity. The 1F3 spot was active against both S. aureus and C. albicans, whereas the 5F1 spot was active against S. aureus only. IR spectral analysis indicated that 5F1 spot to be a triterpenoid. Using IR spectral analysis and an IR library search, the 1F3 spot was identified to be a flavone, which may have a hydroxyl group in ring "A" of the flavone nucleus. Our results indicated that the 1F3 and 5F1 spots are potential free-radical scavengers. Both 1F3 and 5F1 exhibited antimicrobial activity. IR spectral analysis coupled with an IR library search indicated 1F3 and 5F1 to be a flavone and a triterpenoid, respectively.

Introduction

Mirabilis jalapa L. (Nyctaginaceae; 紫茉莉根 zǐ mò lì) is a traditional medicine widely used in many parts of the world for the treatment of various diseases. It is a perennial herb, which reaches a height of 50–100 cm from the tuberous root. It produces beautiful flowers that usually open at around 4 o’clock in the afternoon, and hence, its common name is four o'clock plant. It is a popular ornamental plant grown worldwide for the beauty of its flowers (which can be white, pink, yellow, or multicolored), which have a sweet fragrance. Traditionally, the tuberous root of M. jalapa has been used for treatment of carbuncles (a skin infection caused by Staphylococcus aureus).

Phytochemical investigation of the extracts from this plant showed that it is rich in many active compounds including triterpenes, proteins, flavonoids, alkaloids, and steroids. Alanine, alpha-amyrins, arabinose, beta-amyrins, campesterol, daucosterol, and dopamine were the other compounds reported from extracts of this plant. Gas chromatography (GC)/mass spectral analysis of dichloromethane and methanol extracts of M. jalapa tubers indicated that oleic acid and beta-sitosterol, respectively, were the major compounds present. Liquid chromatography/mass spectroscopy analysis of the aqueous extract of the tuberous root of the plant showed a high content of flavonols. Phenolic acids such as ferulic acid and caffeic acid were also detected in the plant extracts. It has been also reported that the water extract of M. jalapa tubers, containing higher amounts of flavonoids, exhibits antimicrobial and antioxidant activities. In comparison with terpenoid and flavonoid fractions from other parts of the plant, better antioxidant capacity and antimicrobial activity of these fractions from the tuberous root of M. jalapa have been reported. Thus, it was observed that most studies were carried out using a crude extract of a particular solvent or a fraction to determine either various functional activities or the content of major compounds. In this investigation, we aimed to isolate and characterize bioactive components from M. jalapa radix.

Materials and methods

Chemicals

Thin-layer chromatography (TLC) silica gel 60 F254, chemicals, and solvents of analytical grade were purchased from Merck, HiMedia, and Fisher Scientific (Mumbai, Maharastra, India).

Plant material

Mirabilis jalapa L. (紫茉莉 zǐ mò lì) was collected from locally grown flower gardens in Tezpur (91°48′E and 26°38′N), Assam. Voucher specimen was authenticated with the help of the Botanical Survey of India, Shillong (No. BSI/ESC/2011/Plant identification/93).

Extraction and fractionation

The tuberous roots collected were thoroughly washed with tap water and finally rinsed with distilled water. The roots were shade dried for 2 weeks and powdered. Approximately 50 g of ground sample was extracted with 500 mL of 80% methanol for 48 hours. Extraction was repeated and the extracts were pooled and filtered through Whatman number 1 filter paper. The filtrate was concentrated under reduced pressure in a rotary vacuum evaporator (RV10 Control, IKA, Germany). The concentrated extract was air dried to a constant weight (16.1% yield) at room temperature.

Approximately 10 g of aqueous methanolic extract of M. jalapa was dissolved in 100 mL of methanol, which was concentrated to one tenth of its volume, and acidified with 10 mL of 2M H2SO4. The acidified extract was extracted three times with chloroform. The pooled chloroform extract was named fraction 1 (F1). The remaining aqueous methanol solution was basified with NH4OH and extracted three times with ethyl acetate. The ethyl acetate extract was named fraction 2 (F2). The remaining basified aqueous methanol was further extracted three times with isoamyl alcohol and this extract was named fraction 3 (F3). The remaining extract was termed as fraction 4 (F4).4, 5

TLC studies of F1 and F3

Phytochemical screening indicated steroids and triterpenoids as the major compounds in F1, and flavonoids as the major compound in F3. F1 and F3 of aqueous methanolic extract were subjected to TLC studies for isolation of potential free-radical scavenging and antimicrobial spots.

Preparation of TLC plates for collection of spots

Silica gel H without CaSO4 was used as the adsorbent for collection of spots in TLC. The TLC plates were prepared and heated for activation in an oven for 30 minutes at 110°C. The precoated TLC plates were also used for observation of spots after collection.

Approximately 10 mg/mL of F1 and F3 in methanol and 5 mg/mL of standard solutions (β-sitosterol, betulinic acid, ursolic acid, oleanolic acid, rutin hydrate, quercetin, kaempferol, and chlorogenic acid) in methanol were prepared for spotting on the prepared TLC plates. The plates were spotted with 5–10 μL of samples (F1 and F3) and standards, and dried with a light stream of air while spotting.

Developing systems

The following six different solvent systems were used as developing systems: hexane:ethyl acetate:methanol (5:3:2); benzene:methanol (45:55); chloroform:methanol:acetic acid:water (5:4:0.2:1); 1-butanol:acetic acid:water (4:1:5); petroleum ether:chloroform:methanol for terpenoids; and chloroform:ethyl acetate:formic acid for flavonoids.5, 6 The plates were air dried and visualized by spraying with vanillin–sulfuric acid spray followed by heating for 5 minutes at 110°C for detection of steroids and triterpenoids. Flavonoids were detected under ultraviolet rays at 365 nm. The relative front (R) of each fraction was calculated. Spots were separated by running on preparative TLC plates in the selected solvent systems and collected by scrapping silica from the TLC plate for further study.

Free-radical scavenging and antimicrobial activities of TLC-separated components

Precoated TLC plates (TLC silica gel 60 F254) were used for detection of free-radical scavenging activity of the collected spots. The petroleum ether:chloroform:methanol solvent system was used for the assay. The plates were developed by spraying with 0.2% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution in methanol, followed by 30-minute incubation in the dark. Yellow or colorless spots against a purple background showed the presence of free-radical scavenging activity of the spots.8, 9 The experiment was conducted in n = 4 for each sample.

Potential free-radical scavenging spots were further tested against S. aureus (MTCC338) and Candida albicans (MTCC854) by agar diffusion method. The presence of a clear zone against a full-grown pathogen plate shows the antimicrobial activity of the spot. The experiment was conducted in n = 4 for each sample.

GC and Fourier transmission infrared spectral analyses and characterization of the active spot

GC (Perkin Elmer, Germany) of the active spot was conducted by applying the following run conditions: temperature, 80°C for 5 minutes; ramp, 5°C/min to 300°C; hold time, 10 minutes; inlet temperature, 320°C; volume injected, 1 μL; split, 50:1; carrier gas, helium; solvent delay, 8 minutes. The dimension of the column was 60.0 m × 250 μm. The runtime was 60 minutes.

Fourier transmission infrared spectra (FTIR) of potential antioxidant spots were recorded on a modified FTIR spectrophotometer (attenuated total reflectance; Nicolet IS5; Thermo Fisher Scientific, Madison, WI, USA) with a scanning speed of 1 cm/s, a number of 32 scans, and a resolution power of 4. Oleanolic acid (pentacyclic triterpenoid) was used as the standard.

Spots having both free-radical scavenging and antimicrobial activities were tested with ethanolic ferric chloride (FeCl3) for flavone/flavonol content: 50 μL of sample dissolved in methanol was taken to which 10 μL of 10% ethanolic FeCl3 was added. Appearance of bluish green colouration shows the presence of flavone/flavonol.

Concentrated sulfuric acid test (hydroxylation of the flavone/flavonol ring)

Approximately 50 μL of sample in methanol was treated with 0.2 mL of concentrated H2SO4, and Shinoda test (flavones/flavonol/flavanone) was carried out. Magnesium ribbons were added to 50 μL of the sample in methanol followed by addition of concentrated H2SO4. Aluminum chloride colorimetric method was performed to detect the presence of keto and hydroxyl groups in the ring of flavone nucleus. The sample was dissolved in water for testing.

Results

The F1 and F3 fractions of Mirabilis jalapa L. radix (紫茉莉根 zǐ mò lì gēn) showed the presence of terpenoids and flavonoid, respectively. Better separation of F1 was found in the solvent system that included petroleum ether, chloroform, and methanol (petroleum ether:chloroform:methanol = 49:50:1). The presence of steroids and terpenoids (pink/purple spots) was detected upon spraying the plates with vanillin–sulfuric acid (Fig. 1A). Five components were collected from semipreparative TLC plates of F1 (Fig. 1A). F3 was separated in the solvent system containing chloroform, ethyl acetate, and formic acid (chloroform:ethyl acetate:formic acid = 5:4:1) and the fraction showed the presence of flavonoids, which was detected on exposure to ultraviolet rays at 365 nm (Fig. 1B). Two components were collected from the F3 fraction. The R of each spot was also calculated and the values are presented in Table 1. Collected spots were subjected to free-radical scavenging activity assay. The fifth spot of F1 (5F1) and the first spot of F3 (1F3) showed free-radical scavenging activity, as indicated by the development of yellow or colorless spots (circled spots in Fig. 2) against a purple background when sprayed with DPPH solution.7, 8 Potential free-radical scavenging spots were further tested against S. aureus and C. albicans (Fig. 3). The 5F1 spot was active against S. aureus, but it was not active against C. albicans. The 1F3 spot showed activity against C. albicans, but it was found to be only partially active against S. aureus (Fig. 3).

Fig. 1

Chromatograms of fractions F1 and F3. β-sitosterol (βSS), ursolic acid (UA), betulinic acid (BA), and oleanolic acid (OA) are the standards for detection of steroids and terpenoids in F1. Rutin (R), quercetin (Q), kaempferol (K), and chlorogenic acid (CA) are the standards for detection of flavonoids in F3. (A) Solvent system for separation of F1 [petroleum ether/chloroform/methanol (49:50:1)]. (B) Solvent system for separation of F3 [chloroform/ethyl acetate/formic acid (5:4:1)]. 3F1 and 4F1 were found to be low in quantity and were visible only when spotted using a larger quantity.

Table 1

Relative front (Rf) of (A) terpenoid, steroid and F1 components in the petroleum ether:chloroform:methanol (49:50:1) solvent system and (B) flavonoids and F3 components in chloroform:ethyl acetate:formic acid (50:40:10) solvent system.

(A)		Rf
Standard used	βSS	0.83
BA	0.348
OA	0.265
UA	0.254
Separated F1 components	1F1	0.955
2F1	0.811
3F1	0.397
4F1	0.311
5F1	0.236

BA = betulinic acid; OA = oleanolic acid; Q = quercetin hydrate; RH = rutin hydrate; UA = ursolic acid; βSS = beta sitosterol.

Fig. 2

Free-radical scavenging activity of the components separated (1F1, 2F1, 3F1, 4F1, and 5F1 from F1 and 1F3 and 2F3 from F3) by thin-layer chromatography. A colorless spot (circle) against the a purple background indicated the activity [spray, 0.2% DPPH; solvent system, petroleum ether:chloroform:methanol (4.8:5:0.2)]. The experiment was conducted in n = 4. DPPH = 2,2-diphenyl-1-picrylhydrazyl.

Fig. 3

Zone of inhibition of 5F1 and 1F3 against (A, B)Staphylococcus aureus and © Candida albicans. The experiment was conducted in n = 4.

Based on free-radical scavenging and antimicrobial activities, the 1F3 spot was chosen for the GC analysis to evaluate its purity. In the GC analysis, the 1F3 spot showed a single sharp peak with a retention time of 55.15 minutes in GC chromatogram (Fig. 4).

Fig. 4

Chromatogram of 1F3 by gas chromatography analysis. A mass detector was used for the detection of compounds. A single peak was detected in the sample, which showed a retention time of 55.15 minutes.

The 5F1 spot was found to be white and soluble in dichloromethane and methanol. The 1F3 spot was brownish yellow, highly amorphous, and soluble in water/methanol. Data on IR spectral transmission bands of the standard (oleanolic acid), 5F1, and 1F3 spots are presented in Table 2 and Fig. 5.

Table 2

IR spectra transmission bands of standard and collected spots (5F1 and 1F3).

Sample/standard	IR bands of the sample (cm–1)
Oleanolic acid (standard)	3426.49, 2941.36, 2869.75, 1690.83, 1466.56, 1387.68, 1362.59, 1183.37, 1032.76, and 997.25
5F1 spot	3386.90, 2918.58, 2848.99, 1725.54, 1630.25, 1560.18, 1510.24, 1460.05, 1444.04, 1422.14, 1363.85, 1280.31, 1207.89, 1159.36, 1133.74, and 1071.71
1F3 spot	3337.80, 2929.38, 1725.25, 1560.02, 1406.67, 1286.88, 1122.28, 1042.19, 1011.42, and 923.29

IR = infrared.

Fig. 5

Infrared spectra of the standard (S), 5F1, and 1F3. The alkyl group stretching bands were found to be present in spots at 2941.83 cm–1 (5F1) and 2918.56 cm–1 (1F3). The ketone group stretching (1725.25 cm–1) was prominent in 1F3, whereas the band of the anhydride C=O group (3–4-membered ring; 1725.54 cm–1 and 1719.11 cm–1) was found to be present in 5F1 and the standard. Oleanolic acid was used as the standard.

Further chemical characterization of the 1F3 spot showed positive results for the Shinoda test and for concentrated H2SO4, AlCl3, and FeCl3 tests. These results indicated the presence of a flavone nucleus, which may have a hydroxyl group at C-5 of the A ring (maximum absorbance at 395 nm and 415–420 nm with the aluminum chloride colorimetric test; Fig. 6).

Fig. 6

Chemical characterization of the 1F3 spot. (A) 1F3 gives a light brown color when treated with concentrated H2SO4, indicating less hydroxylation. It turns orange on treatment with magnesium ribbons followed by the addition of concentrated H2SO4 (Shinoda test), which indicates the presence of flavone. (B) The 1F3 spot fluorescence at 365 nm. (C) FeCl3 test shows bluish green colour for flavone/flavonol.

Discussion

In this study, the F3 fraction of the hydromethanolic extract of Mirabilis jalapa L. radix (紫茉莉根 zǐ mò lì gēn) was selected for isolation of bioactive components based on its antioxidant capacity and antibacterial activity. The F1 fraction was selected because it was detected in good amounts while screening for major phytochemicals. Many studies have also reported terpenoids from the aerial parts and tubers of M. jalapa.1, 2 TLC is a very basic and easy method for separation of phytochemicals. High-performance TLC is an advancement over TLC. However, to achieve proper separation of different phytochemicals, one has to experiment using different solvents and combinations (trial and error) to standardize the TLC developing system suitable for a particular plant extract. We have tried various combinations of different solvents, and better separation of triterpenoids and steroids in F1 was achieved with petroleum ether:chloroform:methanol (49:50:1) compared with hexane:ethyl acetate (1:1). Presence of β-sitosterol was detected in F1 with petroleum ether:chloroform:methanol (4.9:5:0.1) in our investigation, which is in line with earlier reports.1, 3 The β-sitosterol was reported as a major component in the aerial part. Separation of F3 components was achieved using the chloroform:ethyl acetate:formic acid (5:4:1) solvent system. The same solvent system was used to separate phenolic compounds from kombucha beverages. Alkaloids and flavonoids have also been reported from the root of M. jalapa.

TLC-separated spots of F1 and F3 were subjected to free-radical scavenging and antimicrobial screening. The fifth spot of F1 (5F1) and the first spot of F3 (1F3) showed free-radical scavenging activity (Fig. 2), which was in line with the findings of Hajji et al, who reported that M. jalapa radix can be used for the preservation of food stuffs due to its antioxidant and antimicrobial activities. We also tested the effects of 5F1 and 1F3 against S. aureus and C. albicans. Although the 5F1 spot was active against S. aureus in accordance with an earlier report, it was not active against C. albicans (Fig. 3). Various studies have demonstrated the better antioxidant capacity and antimicrobial activity of flavonoid-rich fractions from the tuberous root of M. jalapa compared with fractions from other parts,2, 4 which supports our finding that the 1F3 spot of flavonoid fraction was active against both C. albicans and S. aureus. According to Gogoi et al, although the F1 and F3 fractions were active against these pathogens, F3 showed a lower minimum inhibition concentration and bigger zone of inhibition against C. albicans than F1. The antimicrobial activity of 5F1 and 1F3 followed the same trend as that of the F1 and F3 fractions. However, no increase in activity was seen on the separated spots. Better antimicrobial activity in the F3 fraction than in the separated spot (1F3) might be due to the synergistic effect of other components.

The IR spectra of the 5F1 spot and standard showed similar stretching and bending band patterns at 2918.58 cm–1, 2848.99 cm–1, 1725.54 cm–1, 1630.25 cm–1, 1560.18 cm–1, 1444.04 cm–1 and 2941.36 cm–1, 2869.75 cm–1, 1719.11 cm–1, 1690.83 cm–1, 1466.56 cm–1, respectively. The IR spectra are interpreted by observing the following banding patterns: Stretching bands at 3000–2800 cm–1 indicate the presence of alkyl groups and shoulder peaks of the carbonyl group are seen in the 2850–2750 cm–1 range. A peak of the carbonyl group of the aromatic three- to four-membered ring is observed between 1750 cm–1 and 1700 cm–1. Peaks between 1600 cm–1 and 1400 cm–1 indicate the presence of aromatic C=C groups. By observing these patterns, it can be said that the 5F1 spot may be a triterpenoid having a carbonyl group. The IR spectra of the 1F3 spot indicated that it may be an aromatic compound (2929.38 cm–1 and 1560.02 cm–1) having carbonyl (1725.25 cm–1) and C–CO–C groups (1122.28 cm–1 and 1286.88 cm–1). A search of the IR spectra of 1F3 in the National Institute of Standards and Technology library indicated that it may be a flavone, which is hydroxylated in ring A.

Cultured M. jalapa has been reported to contain 5,7-dihydroxy-2′-methoxy-6-methylisoflavone (2′-O-methylabronisoflavone) as one of the components showing inhibitory activity against C. albicans. 4′-Hydroxy-2,3-dihydroflavone-7-β-d-glucopyranoside was isolated from the root of M. jalapa. Flavone (1F3) was identified as one of the components of M. jalapa tuberous root in this study. Flavones are phenolic structures containing one carbonyl group. The site(s) and number of hydroxyl groups on the phenol group are thought to be related to their relative toxicity to microorganisms. Existing evidence suggests that increased hydroxylation results in increased toxicity owing to the formation of intramolecular hydrogen bridges, which increase the lipophilic character, and thus, allows for easier penetration through the cell wall.15, 16 The hydrophilicity (free hydroxyl group) of kaurene diterpenoids was increased by adding a methyl group. This modification in kaurene drastically reduced their antimicrobial activity. Thus, in comparison with the terpenic/steroid component (5F1) of F1, the flavone molecule (1F3) of F3 showed better free-radical scavenging and antimicrobial activities, which may be due to the presence of the free hydroxyl group.

Conclusion

Our results indicated that 5F1 and 1F3 spots are potential free-radical scavengers. 1F3 exhibited both antibacterial and antifungal activities, but 5F1 was active only against bacteria. The IR spectral analysis coupled with the IR library search indicated 1F3 and 5F1 to be a flavone and a triterpenoid, respectively. Further studies on 1F3 may be taken up to explore the possible use of these compounds in the treatment of skin afflictions caused by S. aureus and other pathogens.

Conflicts of interest

The authors declare that they have no competing interests.