Pharmacological Studies on Flowers of Ceropegia bulbosa Roxb. var. bulbosa and lushii (Grah.) Hook. F from Thar Desert of Rajasthan, India

 

Sunita Arora*, Sonam Meena¹

^*Professor, Department of Botany, Jai Narain Vyas University, Jodhpur (Raj.), India

¹Research Scholar, Department of Botany, Jai Narain Vyas University, Jodhpur (Raj.), India

 

ABSTRACT:

Objective: Plants synthesize organic compounds continuously to play an important role in maintenance of human health. The characterization of floral chemical constituents of Ceropegia bulbosa Roxb. var. bulbosa and var. lushii was done using GC-MS technique. Methods: Flowers were collected from typical xeric conditions of Indian Thar Desert of Rajasthan, during the month of August-October. The phytochemical compounds were investigated using Perkin-Elmer gas chromatography-mass spectrometry, while the mass spectra of the compounds found in the extract were matched with the National Institute of Standards and Technology (NIST) and Willey 2008 library. Results: Maximum % area is found for 24-Norursa-3,12-diene, is present in maximum amount (41.94%) with retention time (RT) =43.658 minutes, followed by Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (8.72%) with RT=28.029 minutes in the methanolic extract. Pentacosane is present in   maximum amount (39.52%) with retention time (RT)=26.585 minutes, followed by Tetracontane (34.28%) with RT=28.656 minutes in the chloroform extract of flowers of Ceropegia bulbosa Roxb. var. bulbosa. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is present in maximum amount (26.25%) with retention time (RT) =28.039 minutes, followed by Octadecanoic acid, 2,3-dihydroxypropyl ester (12.39%) with RT=30.181 minutes in the methanolic extract. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is present in   maximum amount (40.39%) with retention time (RT)=28.139 minutes, followed by Octadecanoic acid, 2,3-dihydroxypropyl ester (24.49%) with RT=30.237 minutes in the chloroform extract of flowers of Ceropegia bulbosa Roxb. var. lushii. Conclusion: The floral extract of Ceropegia species using methanol and chloroform as a solvent could isolate individual components from mass spectra of the constituents. The isolated bioactive floral phytocomponents of this plant could play an important role in development of new medicine and noval drugs to prevent and treat various diseases or aliments.

 

 

INTRODUCTION:

Plants are sources of bioactive compounds and they continue to play an important role in human health^[1]. India is the largest producer of medicinal herbs and is appropriately called the botanical garden of the world^[2]. Medicinal plants have occupied an important position in the socio-cultural, development of rural people of India. Plants and leaves are considered as one of the main sources of biologically active compounds, even today compound from plants continue to play a major role in primary health care as therapeutic remedies in many developing countries ^[3]. Herbal medicines are safer than synthetic medicines because the phytochemicals in the plant extract target the biochemical pathway. Medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate^[4]. Kumarasingha etal.,^[5] reported that natural compounds from plants provide a prospect in the search for new drugs which are effective, safe and with better pharmacological action than the synthetic drugs. Several compounds, found and isolated from various plants have shown biological properties such as anticancer, anthelmintic, analgesic, antibacterial, anti-inflammatory, antiviral and many other to a lesser or greater extent^[6,7]. The bioactive constituents of medicinal plants can be extracted with different methods and then subjected to evaluation. There are reports of significant differences in physiological activities of plant extracts which depends upon the extraction methods and it emphasizes the importance of choosing an appropriate extraction method for a specific purpose^[8]. The phytochemicals in plants greatly determine the antioxidant, antimicrobial and anti-inflammatory capacity primarily due to their redox properties ^[9] which are important in adsorbing and scavenging of free radicals ^[10].

 

Ceropegia bulbosa Roxb. (Asclepiadaceae) is a threatened medicinal plant distributed in various habitats in semi-arid region of Thar Desert of Rajasthan, India ^[11]. They are usally tuberiferous erect herb and climber, generally grows in comparatively drier parts of the country, shows slight succulence and featured C₄ photosynthesis ^[12]. Ceropegia bulbosa Roxb. var. bulbosa and var. lushii (Grah.) Hook.f. is perennial, twining, herb of sandy substratum, bearing tubers, needs support of other xerophytic bushes/ shrubs. Two varieties are found locally, the broad leaved variety is known as Ceropegia bulbosa var. bulbosa and the variety having thin and long leaves is known as Ceropegia bulbosa var. lushii ^[13]. Tuberous root of the plant is edible and contains a pyridine alkaloid cerpegin ^[14]. Previous reports revealed that it cures cough and cold ^[15], gastrointestinal problems ^[16], Scorpion bite, fertility and viability ^[17], diarrhea and dysentery ^[18] and urinary disorders ^[19,20,21].

 

The plants based phytochemicals are chemical compounds formed during the normal metabolic processes. These chemicals are often referred to as “Secondary metabolites”^[22]. The secondary metabolites are a significant source with a variety of structural arrangements and properties^[23]. In recent years Gas Chromatography- Mass Spectrometry (GC-MS) studies have been increasingly applied to analyze secondary metabolites present in the medicinal plants as this technique has been proved to be a best valuable method for the analysis of essential oil, alcohols, acids, esters, alkaloids, steroids, amino, nitro compounds etc^.[24,25]. Interpretation on mass spectrum was conducted using the database of National Institute Standard and Technology (NIST) having more than 62,000 patterns. The spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library ^[26]. In present study methanol and chloroform extract of flowers of both variety of Ceropegia bulbosa were analyzed by using GC-MS technique to isolate and characterize the bioactive phytochemical constituents from flowers.

 

MATERIAL AND METHODS:

Collection and extraction of plant material:

Fresh flowers of Ceropegia bulbosa Roxb. var. bulbosa and var. lushii. were collected from typical xeric condition of Indian Thar Desert of Rajasthan, during  the month of August-October. The specimen authentication and botanical identification was done by Botanical Survey of India, Jodhpur, Rajasthan. The flowers were thoroughly washed with tap water followed by distilled water, dried under shade for 20 days and ground into fine powder. After sieving the powder was transferred to airtight polyethylene zipper bags, labeled and stored till further use. 2g of coarse powder was transferred to round bottom flask. 200 ml of solvent was added to each flask containing crude powerded plant material. Hot extraction method using soxhlet apparatus was followed with methanol and chloroform as solvent. The solution was then boiled at 60-70º C for 18 hours on water bath, filtered, evaporated to dryness, & final residue was then subjected to GC-MS analysis that was performed at Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New Delhi, India using a GC-MS model; QP 2010 ultra-series, Shimadzu, Tokyo (Japan), equipped with thermal desorption system TD 20. Injection Mode: Split, Flow Control Mode: Linear Velocity, Pressure: 81.9 kPa, Linear Velocity:40.5 cm/sec, Purge Flow: 3.0 mL/min, Split Ratio: 50.0. For GC-MS detection [GC-2010], electron ionization system with ionizing energy of 70ev was used. Helium gas (99.99%) was used as the carrier gas at constant flow rate of 1ml/min in the split mode (10:1) and an injection volume of 2 μl of chloroform solution of different plant part sample was injected into the column with the injector temperature 260ºC, ion-source temperature 230ºC. column oven temperature was maintained at 80 ºC, Pressure was maintained at 81.9 kPa. Equilibrium time was 0.5 min. start m/z ratio was 40.00 and it ended at 650.00. The relative % amount of each component was calculated by comparing its average peak area to the total area, software adopted to handle mass spectra and chromatograms was a Turbomass. The relative percentage of the each extract constituents was expressed as percentage with peak area.

 

RESULT AND DISCUSSION:

Herbal medicine represents one of the most important fields of traditional medicine worldwide. Various extracts from traditional medicinal plants have been tested to identify the source of the therapeutic effects. Significance of employing bioactive compounds in pharmacy to produce drugs for the treatment of many diseases requires purification of compounds ^[27]. Ceropegia bulbosa Roxb. is medicinally more important but it is endangered ^[28,29].

 

GC-MS chromatogram of the methanolic and chloroform extract of flowers of Ceropegia bulbosa Roxb. var. bulbosa showed 66 and 42 peaks (Fig. 1) indicating the presence of 31 and 11 compounds respectively and  flowers of Ceropegia bulbosa Roxb. var. lushii showed 39 and 35 peaks (Fig. 2) indicating the presence of 25 and 15 compounds respectively. Confirmation of their presence was based on retention time (RT), peak area, molecular formula, concentration (%), and molecular weight (Tables 1,2,3 & 4).

 

24-Norursa-3,12-diene is present in maximum amount (41.94%) with retention time (RT) =43.658 minutes, followed by Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester (8.72%) with RT=28.029 minutes and L-Proline, Ethylester is present in minimum amount (0.07%) with RT=16.510 minutes followed by 9,12-Octadecadienoic acid (Z,Z)- (0.10%) with RT=23.617 minutes in the methanolic extract. Pentacosane is present in   maximum amount (39.52%) with retention time (RT)=26.585 minutes, followed by Tetracontane (34.28%) with RT=28.656 minutes and 1-Butanol, 3-methyl-, carbonate (2:1) is present in minimum amount (0.10%) with RT=30.480 minutes, followed by Dodecanoic acid, 1,1-dimethylpropyl ester (0.16%) with RT= 31.217 minutes in the chloroform extract of flowers of Ceropegia bulbosa Roxb. var. bulbosa.

 

Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is present in maximum amount (26.25%) with retention time (RT) =28.039 minutes, followed by Octadecanoic acid, 2,3-dihydroxypropyl ester (12.39%) with RT=30.181 minutes and Glycidyl palmitate is present in minimum amount (0.17%) with RT=25.422 minutes, followed by 2-Dodecanone (0.21%) with RT=11.775 minutes in the methanolic extract. Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester is present in   maximum amount (40.39%) with retention time (RT)=28.139 minutes, followed by Octadecanoic acid, 2,3-dihydroxypropyl ester (24.49%) with RT=30.237 minutes and 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester is present in minimum amount (0.16%) with RT=20.317 minutes, followed by 9-Octadecenamide (0.17%) with RT=30.665 minutes in the chloroform extract of flowers of Ceropegia bulbosa Roxb. var. lushii.

 

The gas chromatogram shows the relative concentrations of various compounds getting eluted as a function of retention time. The peak indicates relative concentration of the components present in the extract. The mass spectrometer analyzes the compounds eluted at different time that identify the nature and structure of the compounds. The large fraction gets dissected into smaller compounds giving rise to appearance of peaks at different m/z (mass to charge) ratio. These mass spectra are fingerprint of that compounds which can be identified from the data library. Methanol is proved to be better solvents as compared to chloroform as it could extract more biologically active compounds. Potent solvents are always better tools for perfect and synchronized extraction.

 

 

 

 

Figure 1. GC-MS chromatogram of the flower extract of Ceropegia bulbosa Roxb. var. bulbosa in (a) methanol and (b) chloform

 

Figure 2. GC-MS chromatogram of the flower extract of Ceropegia bulbosa Roxb. var. lushii in (a) methanol and (b) chloform

 

Table 1. Bio-active compounds in the methanolic extract of flower of Ceropegia bulosa Roxb. var. bulbosa

S. No.	RT (min)	Compound Name	Area  %	Biological Activity
1	9.931	Naphthalene	1.38	Antiseptic, Carcinogenic
2	11.772	2- Undecanone	0.17	Animicrobial activity
3	13.477	1-Tetradecene	1.98	Anti-tuberculosis
4	13.607	Tetratetracontane	0.26	Hypoglycaemic, Antioxidant
5	16.556	Diethyl phthalate	0.32	Estrogenic activity, Antimicrobial, Plasticizer, Antioxidant
6	16.620	1-Hexadecene	2.62	Antibacterial, antifungal, antioxidant
7	16.731	Hexadecane	0.14	Antifungal, Antibacterial and antioxidant activities
8	17.254	Benzophenone	0.35	Antifungal, Anti-HIV, Antimicrobial, Antioxidant, Antiviral and Cytotoxic
9	17.764	8-Pentadecanone	0.83	Heptatoxic, Demyelination, Conjunctivitis activity
10	18.980	Tetradecanoic acid	0.18	Antioxidant, Cancer preventive, Nematicide, Lybricant, Hypocholesterolemic
11	19.442	1-Octadecene	1.67	Finishing agent, Intermediates, Lubricants and Lubricant additives
12	20.318	1,2-Benzenedicarboxylic acid, bis(2-Methylpropyl)ester	0.74	Antimicrobial, antifouling
13	20.490	8-Octadecanone	1.04	Antimicrobial activity
14	21.137	Hexadecanoic acid, methyl ester	1.36	Antioxidant, hypocholesterolenic, antiandrogenic, flavour, nematicide, hemolytic5-alpha reductaee inhibitor
15	21.525	Dibutyl phthalate	0.37	Antimicrobial, Antifungal, Antimalarial, Plasticizer, Ectoparaciticide
16	21.589	n-Hexadecanoic acid	2.30	Antifungal, Antioxidant, Hypocholesterolemic Nematicide, Anti-Androgenic Flavour, Haemolytic 5-Alphareductase Inhibitor, Potent Antimicrobial Agent, Antimalarial And Antifungal
17	21.997	n-Tetracosanol-1	0.95	Anti-bacterial, Anticancer, Anti-oxidant, lowering cholesterol, inhibiting lipid peroxidation, enhancing immune functions, inhibits platelet aggregation,  cardiovascular
18	22.964	10-Nonadecanone	0.25	Anticancer activity
19	23.168	9,12-Octadecadienoic acid (z,z)-,methyl ester	0.45	Hepatoprotective,anti-histaminic, Antieczemic, Hypocholesterolemic
20	23.550	Methyl stearate	0.78	Anti-diarrheal, cytotoxic and antiproliferative  activity
21	23.617	9,12-Octadecadienoic acid (Z,Z)-	0.10	Cancer preventive, Insectifuge, Anti-inflammatory, Nematicide, Hepatoprotective, Antihistaminic, Anticane, Antiarthritic, Antieczemic
22	23.689	Cis-9-Hexadecenal	0.30	Antimicrobial
23	23.959	Octadecanoic acid	0.35	Antibacterial action, Cosmetic, Flavor, Hypocholestrolemic, Lubricant, perfumery, Propecic, Suppository
24	27.747	1H-Indene, 1-hexadecyl-2,3- dihydro-	1.52	Anticancer activity
25	28.029	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	8.72	Antioxidant
26	28.274	1,2-Benzenedicarboxylic acid	0.61	Antioxidant, Antimicrobial, Antifouling activity
27	30.182	Octadecanoic acid, 2,3- dihydroxypropyl ester	3.96	Anticancer, Antimicrobial
28	31.549	1-Heptacosanol	0.42	Nematicidal , anticancer, antioxidant and antimicrobial
29	34.598	Ergost-5-en-3-ol, (3.beta.,24R)-	2.64	Liver disease, jaundice, Artherosclerosis
30	34.883	Stigmasterol	2.78	Antimicrobial activity

 

 

 

 

 

 

Table 2. Bio-active compounds in the chloroform extract of flowerof Ceropegia bulosa Roxb. var. bulbosa

S. No.	RT (min)	Compound Name	Area  %	Biological Activity
1.	20.928	7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione	2.03	Antimicrobial activity
2.	21.586	n-Hexadecanoic acid	0.97	Antifungal, Antioxidant, Hypocholesterolemic Nematicide, Anti-Androgenic Flavour, Haemolytic 5-Alphareductase Inhibitor, Potent Antimicrobial Agent, Antimalarial And Antifungal
3.	23.257	Heneicosane	4.97	Anibacterial, Oviposition attractant pheromone
4.	26.585	Pentacosane	39.52	Antibacterial activity
5.	28.656	Tetracontane	34.28	Anti-inflammatory and Analgesic activity
6.	29.678	Tetratriacontane	0.55	Antibacterial and Antifungal
7.	30.670	9-Octadecenamide	0.57	Good therapeutic agent for the treatment of sleep disorders and pain
8.	32.001	Hexatriacontane	0.41	Free Radical scavenging
9.	33.215	Stigmast-5-en-3-ol, (3.beta.)-	0.55	Anti-inflammatory, Anti-pyretic, Anti-ulcer, Antiarthritic
10.	35.643	.gamma.- Sitosterol	3.28	Hupolipidemic property
11.	39.527	5,11,17,23-Tetratert-butylpentacyclo(19.3.1.1(3,7).1(9,13).1(15,19))octacosa-1(25),3(28),4,6,9(27),10,12,15(26),16,18,21,23-dodecaene-25,26,27,28-tetrol	3.53	Membrane carriers for the transport of chiral amino acids used as extractants, transporters and optical sensors

 

 

 

 

 

Table 3. Bio-active compounds in the methanolic extract of flower of Ceropegia bulosa Roxb. var. lishii

S. No.	RT (min)	Compound Name	Area  %	Biological Activity
1.	9.934	Naphthalene	1.71	Antiseptic, Carcinogenic
2.	13.476	1-Tetradecene	2.45	Anti- tuberculosis activity
3.	16.618	1-Hexadecene	3.12	Antibacterial, Antifungal, Antioxidant
4.	17.762	8-Pentadecanone	0.82	Heptatoxic, Demyelination, Conjunctivitis activity
5.	19.439	1-Octadecene	1.89	Finishing agent, Intermediates, Lubricants and Lubricant additives
6.	20.316	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	0.65	Antimicrobial activity, alpha- Glucosidase inhibition and the in vitro hypoglycemic effect
7.	20.488	8-Octadecanone	0.92	Antimicrobial activity
8.	21.136	Hexadecanoic acid, methyl ester	6.98	Antibacterial and Antifungal, Antioxidant Hypocholesterolemic, Nematicide, Insecticide Lubricant, Antiandrogenic Flavor, Hemolytic
9.	21.521	Dibutyl phthalate	0.61	Antifungal, antimicrobial, anti-malarial, plasticizer and ectoparasiticide
10.	21.591	n-Hexadecanoic acid	0.61	Antifungal, Antioxidant, Hypocholesterolemic Nematicide, Anti-Androgenic Flavour, Haemolytic 5-Alphareductase Inhibitor, Potent Antimicrobial Agent, Antimalarial And Antifungal
11.	22.960	10-Nonadecanone	0.26	Anticancer activity
12.	23.164	9,12-Octadecadienoic acid (z,z)-, methyl ester	0.81	Hepatoprotective,anti-histaminic, Antieczemic, Hypocholesterolemic,
13.	23.548	Methyl stearate	4.20	Anti-diarrheal, cytotoxic and antiproliferative  activity
14.	27.745	1H-Indene, 1-hexadecyl-2,3- dihydro-	5.02	Anticancer activity
15.	28.039	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	26.25	Antioxidant
16.	28.272	1,2-Benzenedicarboxylic acid	1.41	Antioxidant, Antimicrobial, Antifouling activity
17.	29.062	Docosane	0.25	Antibacterial activity
18.	29.785	1H-Indole-3-Ethanamine	1.78	Used in Neurotransmitter & psychedelics
19.	30.181	Octadecanoic acid, 2,3- dihydroxypropyl ester	12.39	Anticancer, Antimicrobial
20.	30.807	Tetracontane	0.37	Anti-inflammatory and Analgesic activity
21.	34.591	Ergost-5-en-3-ol, (3.beta.,24R)-	1.54	Liver disease, jaundice, Artherosclerosis
22.	34.882	Stigmasterol	1.76	Antimicrobial activity
23.	35.632	.gamma.- Sitosterol	3.54	Hupolipidemic property
24.	43.186	Methyl commate A	7.98	Anthelmintic effect

 

 

 

 

 

Table 4. Bio-active compounds in the chloroform extract of flower of Ceropegia bulosa Roxb. var. lishii

S. No.	RT (min)	Compound Name	Area %	Biological Activity
1	20.317	1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester	0.16	Antimicrobial activity, alpha- Glucosidase inhibition and the in vitro hypoglycemic effect
2	20.933	7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione	1.47	Antimicrobial activity
3	21.599	I-(+)-Ascorbic acid 2,6- dihexadecanoate	0.29	Anti-pigmentation effect
4	23.257	Heneicosane	1.63	Anibacterial, Oviposition attractant pheromone
5	26.585	Pentacosane	13.20	Antibacterial activity
6	28.139	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester	40.39	Antioxidant
7	28.304	Bis (2-ethyl hexyl) phthalate	0.99	Antimicrobial, Plasticizer
8	29.680	Tetratriacontane	0.29	Antibacterial and Antifungal
9	30.237	Octadecanoic acid, 2,3- dihydroxypropyl ester	24.49	Anticancer, Antimicrobial
10	30.529	Tetratetracontane	0.35	Antioxidant, Hypoglycemic
11	30.665	9-Octadecenamide	0.17	Good therapeutic agent for the treatment of sleep disorders and pain
12	30.816	Tetracontane	8.81	Anti-inflammatory and Analgesic activity
13	32.002	Hexatriacontane	0.30	Free Radical scavenging
14	35.645	.gamma.- Sitosterol	0.57	Hupolipidemic property
15	39.535	5,11,17,23-Tetratert-butylpentacyclo(19.3.1.1(3,7).1(9,13).1(15,19))octacosa-1(25),3(28),4,6,9(27),10,12,15(26),16,18,21,23-dodecaene-25,26,27,28-tetrol	3.58	Membrane carriers for the transport of chiral amino acids used as extractants, transporters and optical sensors

 

 

 

CONCLUSION:

GC-MS technique is a fast and direct analytical approach for isolation and identification of bioactive compounds and only few grams of plant material is required. The technique of fingerprint could really identify the false herbal products. The construction of chromatographic fingerprints aims at evaluating the quality of herbal medicines ^[30].We are first to report the presence of some important bioactive chemical constituents from flowers of this plant using GC-MS technique. Extraction of valuable biological compounds from flowers can open new era for pharmaceutics and drug design companies. Apart from this other trials are required before practical applications and the plant also need proper conservation strategies as it is being endangered.

 

ACKNOWLEDGEMENT:

Authors express sincere thanks to Prof. P.K. Kasera for providing academic support and the CAS Department of Botany J.N.V. University, Jodhpur (Rajasthan) for providing research facilities, infrastructure and technical support. I am also thankful to University Grant Commission (UGC), New Delhi for providing financial support.

 

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Received on 04.03.2018        Modified on 13.04.2018

Accepted on 10.05.2018       ©A&V Publications All right reserved