Development and Screening of Anticonvulsant Polyherbal Formulation

 

Mahanthesh M.C.¹*, Girendra Gautam², Jalalpure S.S.³

¹Bhagwant University, Ajmer

¹Tatyasaheb Kore College of Pharmacy, Warananagar-416113, Maharashtra, India,

²Department of Pharmacy, Bhagwant University, Ajmer, Rajasthan, India

³Department of Pharmacognosy, KLE’S College of Pharmacy and Dr. Prabhakar Kore Basic Science Research Centre, KLE University, Belgaum-10, Karnataka, India,

 

ABSTRACT:

Objective:  To explore the pharmacognostical characteristics and in vivo anticonvulsant activity of chloroform extracts of Diplocyclos palmatue leaves, Abutilon indicum Linn sweet stem and Cassia occidentalis whole plant formulation. Methods: The plants were successively extracted using chloroform. The extract was screened for phytochemicals using HPTLC, GCMS techniques. The extract was also screened for acute toxicity and herbal formulation anticonvulsant activity, against MES and PTZ induced convulsions, using Wistar albino rats. Results: The phytochemical screening study reveals the presence of more chemical constituents in chloroform extract. We found no significant changes in average body weight of animals, up to tested oral dose of 3000 mg/kg, during acute toxicity study and suspension having good stability on storage. The in vivo study reveals the anticonvulsant activity of chloroform extract against MES and PTZ induced convulsions. The chloroform extract is found to be more potent, similar to Phenytoin, in controlling both MES and PTZ induced convulsions. Conclusions: The results obtained suggest that the formulation has remarkable anticonvulsant activity. Also, our study indicates the potential application of these combined herbal dosage forms could be suitable for anticonvulsant activity. However, this claim demands through investigation in other models for anticonvulsant activity.

 

 

 

 

 

INTRODUCTION:

The use of herbal medicinal products (HMPs) has several folds increased over the past few decades. The general public’s use of herbals has increased over the past 10 years and is relatively economical, provides solution where other therapies do not, one of the major beliefs that HMPs are always safe.

 

Aside from the need to appraise these products for safety and efficacy, health care providers and the public need to know whether interactions might occur when these products are used in combination with conventional drugs.¹ A mental or neurological disorder encompasses broad range of conditions that result in dysfunction of brain, spinal cord and nerves.² Approximately 1% of the world’s population has epilepsy, the second most neurologic disorder after stroke.³ Epilepsy, which has been described as a chronic disorder of the central nervous system of various etiologies, characterized by recurrent seizures due to excessive discharge of cerebral neurons is a major medical and social problem.⁴The treatments of epilepsy still remains inadequate even though new anticonvulsants are being developed. Furthermore, most of the marketed drugs used as antiepileptic agents have serious side effects; and even life-threatening conditions are experiencing by the epilepsy patients.⁵

 

Figure 1: (A) Diplocyclos palmatus (DP), (B) Abutilon indicum (AI) and (C) Cassia occidentalis (CO)

 

Diplocyclos palmatus (DP) Linn. leaves (Fig 1(A)) is an annual climbing herb.⁶, slender much branched tendril climber and is distributed throughout India on hedges and bushes up to 1,200 m from a thick permanent root stock, tendrils 2-fid; leaves simple, alternate, membranous, 5-lobed, scabrid above, cordate at base, flower yellow. The traditionally various parts used in thermogenic, anti-inflammatory, tonic, skin disease, paralysis of tongue, snakebite⁷ and are reported to posses various pharmacological actions such as antitumor and antioxidant⁸, sexual behavior of male rats⁹, anti-arthritic activity.¹⁰ The cytotoxicity¹¹, sex selection drug¹², anthelmentic¹³, antidiabetics¹⁴ and jaundice¹⁵, anti-microbial¹⁶, anti hyperlipidemic.¹⁷

 

Abutilon indicum (AI) (Fig 1(B)) is an herbaceous woody perennial, softly tomantose shrub¹⁸, found in hotter parts of India. Found as a weed in the sub Himalayan tract and other hills up to 1.200M¹⁹ and up to 3 m in height. The leaves are evergreen, base-cordate, stipulate, fili form, ovate, acuminate, toothed, and rarely subtrilobate and 1.9-2.5 cm long. Petiole 1.5-1.70cm long, cylindrical, yellowish in colour, stellate and hairy. The flowers are yellow in color, peduncle jointed above the middle. The petioles are 3.8-7.5 cm long; stipules 9 mm long; pedicels often 2.5-5mm long, axillary solitary, jointed very near to top and the seeds are 3-5mm, kidney shaped, reniform, tubercled or minutely stellatehairy, black or dark brown. Traditionally various parts used in anthelmentic, antiemetic, anti-inflammatory, in urinary or uterine discharge, piles and antidote. It is used in treatment of fever, dry cough; bronchitis, gonorrhea and leprosy and aphrodisiac.²⁰ Root is demulcent, diuretic, nervine tonic, fever, and arthritis. Leaves are used in bleeding piles, diuretic, demulcent and toothache. Flowers are applied in boils and ulcers. Seeds are expectorant, aphrodisiac, laxative, gonorrhea. Bark is astringent and diuretic, decoction of leaves is used as an eye-wash and mouth wash in toothache²¹ and are reported to posses various pharmacological action such as bronchial asthma²², hepatoprotective, male contraceptive and analgesic.²³ The larvicidal, pupal deformities²⁴, immunostimulating²⁵ , anti-arthritic activity²⁶, cytotoxic and antimicrobial activity²⁷.The  hepatotoxicity²⁸, anti-anxiety²⁹ and seed powder on genital organs and fertility of female³⁰, glucose absorption and stimulates insulin³¹.The diuretic³², antisnake venom³³, hepatoprotective³⁴ and also antimycotic³⁵, arthritic³⁶.

 

Cassia occidentalis (CO) (Fig 1(C)) whole plant is found in throughout India, it grows abundantly on wastelands immediately after the rains. A diffuse offensively odorous under shrub with furrowed sub glabrous branches, leaflets 3-5 pairs, flowers yellow, in short peduncled few flowered racemes, fruits cylindrical or compressed, transversely septate glabrous pods containing 20-30 seeds ovoid, smooth and shiny dark olive green or pale brown, scattered from the Himalayas to the Western Bengal, South India, Burma and Ceylon.³⁷ In India up to an altitude of 1,500 m. traditionally various parts  used in expectorant, diuretic, leaves used internally and externally in scabies, ringworm and other skin disease, bronchitis, constipation, fever, epilepsy and convulsions ^38.39,40, anti-allergic, anti-inflammatory and anti-oxidant ⁴¹and are reported to posses various pharmacological action such as Toxicological reproductive⁴², antidote of poison, blood purifier, expectorant, anti-inflammatory agent, liver disease⁴³ and also in Unani system of medicine melanoblast cell line⁴⁴, relaxant.⁴⁵ The anti-nociceptive⁴⁶, skin diseases like psoriasis; leprosy⁴⁷, oxidative stress⁴⁸ and antifungal, anti-diabetic⁴⁹. The hepato-protective⁵⁰, cytotoxicity, antibacterial agent⁵¹, antimalarial⁵², antianxity and also in antidepressant⁵³, nephroprotective⁵⁴. The neutron activation analysis.⁵⁵ Natural products have been our single most successful source of medicines. Each plant is like factory capable of synthesizing unlimited number of highly complex and unusual chemical substances whose structures could otherwise escape the imagination forever⁵⁶. Moreover, some products are more effective in a liquid form and are used commonly by young children’s or the elderly to overcome problem of swallowing the solid oral dosage forms. Most of the orally administered Ayurvedic formulations belong to liquid form of drug or drug combination.⁵⁷ The literature survey reveals the use of DP leaves, AI stem and CO whole plant in traditional system of medicines to treat convulsions.⁵⁸ the empathy of polyhedral formulation is well documented in the ancient literature. Compared to the single herb, the polyherbal formulation has better and extended therapeutic potential. Hence, the present study was planned to formulate and to evaluate the anti-convulsion potential with well-known antiepileptic drug Phenytoin as standard by using Maximal Electroshock (MES) and PTZ induced convulsions.

 

MATERIALS AND METHODS:

Collection and Authentification of Plant Material:

In present study, the DP leaves, AI stem and CO whole plant was collected from waste place surrounding area of modala vittalapura, Shimoga (District), Karnataka. The whole plant  was authenticated from botanist Dr. S.R. Yadav, Head of  the Department, Department of botany Shivaji University, Kolhapur, Maharashtra, India.( University TKCP/SU/BOT/ 164/2011). After authentification, the drug were dried at room temperature, until they were free from the moisture and subjected to physical evaluation with different parameters.

 

Preparation of Extract:

The shade-dried DP leaves, AI stem and CO whole plant were reduced to fine powder (# 40 size mesh) and around 300gms of powder was subjected to successive hot continuous extraction (soxhlet) with petroleum ether, chloroform and ethanol. Finally the drug was macerated with chloroform-water. Each time before extracting with the next solvent the powdered material was air dried in hot air oven below 50 ⁰ C. After the effective extraction, the solvents were redistilled, the extract was then concentrated on water bath and the extract obtained with each solvent was weighed. Its percentage was calculated in terms of air-dried weight of plant material. The colour and consistency of the extracts was noted. The chloroform extract was taken for phytochemicals screening.

 

Photochemical Screening:

The presence of various phytoconstituents viz alkaloids, carbohydrates, glycosides, flavonoids, triterpinoids, phenolic, steroids and proteins , aminoacids, fats, fixed oil and saponins were determined using suitable chemical test.⁵⁹

 

TLC Analysis:

Analytical TLC plates were prepared by pouring the silica gel G (TLC-grade; Merck India) slurry on the glass plates. Thin layer plates were dried for 30 minutes in air and then in an oven at 110°C for another 30 minutes. For qualitative exertion, the chloroform extract were spotted, in duplicate, on TLC plate, about 2cm above from the bottom, by using capillary tubes. The range of sample volume was controlled, spreading not more than 0.5cm. Three different solvent systems were used as developing systems. CE- EA: M: W (100:13.5:10), CE- LPE: EA: FA (75:25:1) and CE- nB: EA: GAA (40:40:29:1). The standard abbreviations used are: CE= chloroform extract, EA= ethyl acetate, M= methanol and W= water, LPE= light pet ether, FA= formic acid, nB= n-butenol and GAA= glacial acetic acid. The plates were placed in previously saturated TLC chamber with mobile phase. The plates were developed in respective mobile phase up to 80%, dried and spots were visualized by exposing the plates to iodine vapor.⁶⁰

 

FT-IR Analysis:

 The IR spectrum for chloroform of DP leaves, AI stem and CO whole plant was obtained using FT-IR Agilent-630 over the frequency range from 4000-650 cm^-1. The spectra were plotted against Wave number cm^-1 Vs Transmittance (%).⁶¹

 

HPTLC Fingerprint:

CAMAG HPTLC system equipped with Linomat 5 applicator, TLC scanner-3, repro star 3 with 12 bit CCD camera for photo documentation, controlled by WinCATS-4 software was used. All solvents used for HPTLC analysis were purchased from MERCK. 100 mg of extract was dissolved in 5ml of methanol and used, as tests solution, for HPTLC analysis. 10µl samples were spotted in the form of bands of width 8mm with a Camag microlitre syringe on precoated silica gel glass plate 60F-254. The distance between two tracks was 10 mm. An application volume of 10μl was applied at a position of 10mm from the bottom on the plate and the length of chromatogram run was 80mm from the application position.  The sample loaded plate was kept in TLC twin trough developing chamber (after saturation with solvent vapor for 30 minutes) containing mobile phase. The ethyl acetate: methanol: water (3:5:8), Light pet. Ether: ethyl acetate: formic acid (3:5:8) and n-butanol:ethyl acetate: GAA (3:5:8) were employed as mobile phases. Aforesaid mobile phases were used for analysis, the chloroform extract of DP leaves, AI stem and CO whole plant. The linear ascending development was carried out in 20cm x 10cm twin trough glass chamber saturated with the mobile phase. The developed plate was dried by hot air to evaporate solvents from the plate and the plate was fixed in scanner stage and scanning was done at 254nm. Densitometric scanning was performed on CAMAG TLC Scanner-3 and operated by CATS software (V 4.63, Camag). The scanner converts bands into peaks, peak height and area which were related to the concentration of the substance on the spot.⁶²

 

GC-MS Analysis:

GC-MS analysis of the extracts was performed with Shimadzu system and Gas chromatograph interfaced to a Mass spectrometer (GC-MS, 2010) equipped with a Elite-1 fused silica capillary column (RTZ i 5ms; 30 mm x 0.25 mm 1D). For GC-MS detection, and electron ionization system with ionizing energy of 70eV was used. Helium gas (99.999%) was used as the carrier gas at constant flow rate of 1 ml/min and an injection volume of 2µl was employed (Split ratio of 50:50). The injector temperature of 250°C and ion-source temperature of 250°C was used.  The oven temperature was programmed from 80°C (isothermal for 3 min), with an increase of 10°C/min, to 280°C, ending with a 10 min isothermal at 280°C. Mass spectra were taken at 70eV, a scan interval of 0.5 seconds and fragments from 40 to 550 Da. The total GC running time was 33 min. The relative % amount of each component was calculated by comparing its average peak area to the total areas. The software adopted to handle mass spectra and chromatograms was a turbo mass. Interpretation on mass spectrum of GC-MS was done using the database of National Institute of Standard and Technology (NIST) having more than 62,000 patterns. The mass spectrum of the unknown component was compared with the spectrum of the known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.^{63, 64}

 

Acute Toxicity Study:

The experimental protocol was approved by the Institutional Animal Ethics Committee of Tatyasaheb Kore College of Pharmacy, Warananagar, (Maharashtra), India (Ref. No.IAE/TKCP/2012/11, date: 21/12/2012) and animals were maintained under standard conditions in the animal house approved by Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA). The acute toxicity of the extract was determined by the method of Reed and Meunch ⁶⁵ on Wistar albino rats. 35 Wistar albino rats of either sex, weighing 185-200g were divided into 7 groups, each containing five animals. The rats were fasted for 18 hours, with water and libitum. The animals were administered with solution of chloroform extract of DP leaves, AI stem and CO whole plant in distilled water and Tween 20 mixture.  The aqueous solutions of extracts containing 2% Tween 20 were administered to rats orally. The dose was administered by gavage using a stomach tube. Group 1 was kept as untreated control. Group 2, 3, 4, 5 and 6 were administered orally with a dose of 250, 500, 1000, 2000 and 3000 mg/kg body weight, respectively. Group 7 was given 2% Tween 20 in distilled water and kept as vehicle control. The number of animals dead in each group, after 72 hours of administration of the drug was recorded.⁶⁶

 

Development of Formulation:

Formulations are the best media to convey the medicament to bodily organs it is also proved practiced to formulate the medicament into the formulation so as to perceive the intended result in an effective manner.  Fresh air dried powdered crude drugs of each plant were extracted with chloroform by adopting soxhlet extraction procedure. The extract were filtered and concentrated to dryness at room temperature to avoid the decomposition of the natural metabolites. Extract were suspended in water using tween 20 as suspending agent, potassium sorbet as preservative and sodium CMC, soya lecithin as stabilizer. The extracts were mixed in the same concentration of (1:1:1).

 

Pharmaceutical Evaluation:

Organoleptic characteristics: Organoleptic characteristics such as odour, taste and colour of formulation were evaluated after 1h of preparation at room temperature.  Sedimentation volumes: Sedimentation volumes of the suspension were evaluated after 1h of preparation at room temperature. Fifty milliliter of suspension was taken in a 50mL stoppered graduated measurinf cylinder. The suspension was thoroughly dispersed by inverting the vessel three times and it was allowed to settle for 3min. the sediment volume was recorded as the original sediment volume (Ho).The cylinder was kept undisturbed for 1h, and the sediment volume was then measured. This was considered as the final sediment volume (Hu).The sedimentation volume was calculated using the following equation.^{67, 68, 69}

                                         H₀

sedimentation volume =--------------

                                         H_u

 

Determination of redispersibility:

The redispersibility of suspension was evaluated after 1h of preparation at room temperature. It was measured by placing suspension (30mL) in a 100mL graduated cylinder. After storage and sedimentation, the cylinder was rotated through 360° at 20rpm. The end point was recorded when the base of the cylinder was clear of sediment and the suspension was uniform. Determination of pH: The pH of fresh suspensions was measured at room temperature after 1h of preparation using a digital type pH meter (Merck Digital).⁷⁰ Accelerated stability study: Accelerated stability studies were carried out for formulations at the temperature of 8°c and 45°c in electrically heated and thermostatically controlled ovens.^{71, 72} The stability was studied over a period of 3 months. Different parameters such as the colour, odour, pH, redispersibility, and sedimentation volumes were recorded for all the formulations after 90 days of storage.

 

Screening of Anticonvulsant Activity of Herbal formulation:

Anticonvulsant activity will be screened against MES and PTZ induced convulsions on group of six albino rats either sex. The activity will be compared with standard Phenytoin.

 

MES Induced Convulsions:

The maximal electro-shok (MES) is applied through the ear clip electrodes using electroconvulsiometer electric current of 150 mA for 0.2sec, excitation is produced. The MES convulsions are divided into five phases such as (a) tonic flexion, (b) tonic extensor, (c) Clonic convulsion (d) Stupor and (e) recovery or death. A substance is known to posses’ anticonvulsant property if it reduces or abolishes the extensor phase of MES convulsions. Animals were weighed, numbered and divided into three groups, each consisting of 6 rats and one group was used as control (Saline treated) and the other as reference standard (phenytoin treated). Animal was hold properly and placed with ear clip electrode and applied the prescribed current and noted the different stages of convulsions, i.e. a) tonic flexion, b) tonic extensor, c) clonic convulsion d) stupor and e) recovery or death. Noted the time (sec) spent by the animal in each phase of convulsions. Repeated the same for other animals of the control group. The animals were injected with phenytoin intra-peritonially (25mg/kg i.p) and after 30 minutes the animals were subjected to electro convulsions and noted the reduction in time or abolition of tonic extensor phase of MES convulsions. The formulation was tested at a dose of 200 mg/kg orally.

 

PTZ Induced Convulsion:

Pentylene tetrazole is a central nervous system stimulant. It produces jerky type of clonic convulsions in rats. It acts through GABA benzodiazepine receptor mechanisms in brain. Animals were weighed, numbered and divided into three groups each consisting of 6 rats. Group one was used as control (pentylenetetrazol treated; 80 mg/kg) and the group 2 for studying the protective effect of diazepam (diazepam and pentylenetetrazol treated; at dose of 4 and 80 mg/kg, respectively). The control group animals were injected with pentylenetetrazol (80 mg/kg) intra-peritonially and noted the onset of action (indicated by straub’s tail, jerky movements of whole body and convulsions) and severity of convulsion. The second groups of animals were first injected with diazepam intraperitonially. After 30 minutes, the pentylenetetrazol was injected to the same animals and noted either delay or complete abolition of convulsions in rat previously treated with diazepam. The formulation were tested at a dose of 200 mg/kg orally.⁷³

 

RESULTS AND DISCUSSION:

physicochemical analysis of DP leaves, AI stem and CO whole plant course powder and the percentage of chloroform extract, extractive values and the loss on drying (LOD), hot air oven maintained at 105 -110°C. Deterioration time of the plant material depends upon the amount of water present in plant material. If the water content is high, the plant can be easily deteriorated due to fungal attack, to determine the chemical constituents correctly, it is necessary to calculate LOD results are expressed. The drug powder was observed under UV light at 254 nm and 364nm and colour was noted (Supplemental table no. 1).

 

Table 1: Physicochemical analysis of Chloroform for DP leaves, AI stem and CO whole plant

Sr. No	Parameters	Literature	Observation
(A) Chloroform extract of DP leaves
1.	Physical Tests
	Nature	Coarse powder	Coarse powder
	Color	Green  color	Green  color
	Odour	Odourless	Odourless
2.	Extractive value	--	2.103 %
3.	Loss on drying	--	3.45%
4.	Total ash	--	2.76%
5.	Acid insoluble ash	--	1.68%
6.	Water soluble ash	--	1.36%
7.	Fluorescence analysis	--	Dark fluorescence
(B)Chloroform extract of AI stem
1.	Physical Tests
	Nature	Coarse powder	Coarse powder
	Color	Yellowish   color	Yellowish  color
	Odour	Odourless	Odourless
2.	Extractive value	--	2.42 %
3.	Loss on drying	--	3.45%
4.	Total ash	--	2.57 w/w
5.	Acid insoluble ash	--	1.60 w/w
6.	Water soluble ash	--	1.30w/w
7.	Fluorescence analysis	--	Dark fluorescence
(C) Chloroform extract of CO whole plant
1.	Physical Tests
	Nature	Coarse powder	Coarse powder
	Color	Yellowish color	Yellowish color
	Odour	Odourless	Odourless
2.	Extractive value	--	2.62 %
3.	Loss on drying	--	2.38 %
4.	Total ash	--	3.76 %w/w
5.	Acid insoluble ash	--	2.63 % w/w
6.	Water soluble ash	--	1.26 % w/w
7.	Fluorescence analysis	--	Dark fluorescence

 

Table 2: Results of Qualitative Chemical Investigation of Chloroform for DP leaves, AI stem and CO whole plant

Phytochemical name	Chloroform extract
	Diplocyclos palmatus	Abutilon indicum	Cassia occidentalis
Alkaloids	-	-	-
Carbohydrates	+	+	+
Glycosides	+	+	+
Flavonoids,	-	-	-
Triterpinoids	-	-	-
Tannins and Phenolic comp	+	+	+
Steroids	+	+	+
Proteins	+	+	+
Amino Acids	-	-	-
Lipids	-	-	-
Saponin`	+	+	+

+: Present, -: Absent

Phytochemical screening

The phytochemical screening test has been conducted after successive solvent extraction was showed in table no.2, the primary and secondary metabolites in solvent extraction which are helpful in predicting their therapeutic properties and important aspect of the process of establishing herbal medicine quality and in determining the chemical constituents of plant materials.

 

They are also useful in locating the source of pharmacologically active chemical compounds, the preliminary phytochemical analysis revealed the presence of glycosides, flavonoids, triterpinoids, carbohydrates, steroids, phenolic compounds, saponins, amino acids and proteins. Flavonoids, triterpinoids, carbohydrates and glycosides were found to be present in all the extracts.

TLC analysis:

TLC is used as an easier method of initial screening with a semi quantitative evaluation of herbal medicines than with instrumental chromatography. Thin-layer chromatography is a technique in which a solute undergoes distribution between two phases, a stationary phase acting through adsorption and a mobile phase in the form of a liquid. The chloroform extract of DP leaves, AI stem and CO whole plant were subjected to TLC analysis in order to identify the phytochemicals. In the present study, the most suitable TLC system for analysis was shown to be ethyl acetate: methanol: water. The TLC profiling reveals the presence of diverse group of phytochemicals shown in figure 2.

 

 

 

                                        (Ethyl acetate (100)                        Methanol (13.5):                                                 Water (10)

Figure 2: TLC chloroform extract of (A) Diplocyclos palmatus (DP), (B) Abutilon indicum (AI) and (C) Cassia occidentalis (CO)

 

FT-IR analysis:

The IR spectroscopy is one of the analytical techniques which offer the possibility of chemical identification. After absorption of IR radiations, the molecules vibrate and give absorption spectrum. The IR spectrum of chloroform extract of DP leaves, AI stem and CO whole plant figure 3.It is most useful for the identification of purity and gross structural details. The wavelength of light absorbed is characteristic of the chemical bond as can be seen in the annotated spectrum. By interpreting the infrared absorption spectrum, the chemical bonds in a molecule can be determined.

 

Figure 3: IR chloroform extract of (A) Diplocyclos palmatus (DP), (B) Abutilon indicum (AI) and (C) Cassia occidentalis (CO)

The absorption peaks corresponded to different functional groups of phytochemicals present in the chloroform extract of DP leaves, AI stem and CO whole plant in Table 3. The DP exhibited a characteristics band at 2920.44 cm^-1 indicating the presence of (C-H stretching group), 2852.86 cm^-1 (C-H stretching group), 1618.45.cm^-1 for aromatic groups (C=C), 1459.2cm^-1 for aromatic bending, 1378.31cm^-1 for CH bending,1162.43cm^-1 for C-O alcohol stretching.

 

Table 3: IR of Chloroform for DP leaves, AI stem and CO whole plant

(A) Diplocyclos palmatus
Wave length (cm-1)		Interpretation
2920.44		C-H  Alkenes stretching vibration band
2852.86		C-H Alkenes stretching vibration band
1702.03		C-H Alkenes stretching vibration band
827.09		Alkenes asymmetric C-H stretching vibration band
1618.45		Aromatic  C=C Alkenes stretching vibration band
1459.28		C=C Aromatic  bending vibration band
1378.31		C-H  Alkane bending vibration band
1162.43		C-O Alcohol  stretching vibration band
1076.04		C-H Alkane stretching vibration
1028.17		C-H Alkane  stretching vibration
969.17		C=H bending vibration band
(B) Abutilon indicum
2924.60	C-H  Alkane stretching vibration band
2855.45	C-H  Alkane stretching vibration band
1711.74	C=O  Aldehyde stretching vibration band
1459.92	C=C Aromatic bending vibration band
1378.07	C-H stretching vibration band
1274.02	C-O Alcohol  stretching vibration band
1186.37	C-O Ketone vibration band
1081.17	C-O Alcohols stretching vibration band
967.62	C=O Alcohols bending vibration band
(C) Cassia occidentalis
2921.95	Alkenes asymmetric C-H stretching vibration band
2852.67	Alkenes asymmetric C-H stretching vibration band
1709.84	C=O stretching vibration band
1460.07	Alkane-CH2-bending vibration band
1492.29	Alkane-CH2-bending vibration band
1379.71	-NO2 Nitro compounds
1269.96	C-C stretching vibration band
1186.02	C-N Amines
1035.65	C-O Alcohols, ethers, carboxylic acid, esters stretching vibration
1117.90	C-N Amines
1081.11	C-O Alcohols, ethers, carboxylic acid, esters stretching vibration
969.40	Vinyl C=C bending vibration band
665.01	C-H Aromatic rings

 

The 1076.04cm^-1 C-H stretching and 969.17cm^-1 for vinyl (C=H bending) were exhibited by DP and the AI exhibited a characteristics band at 2924.60 cm^-1 indicating the presence of (C-H stretching group), 2855.45 cm^-1 (C-H stretching group),1711.74cm^-1 for aldehyde (C=O), 1459.92cm^-1 for aromatic groups (C=C),1378.07cm^-1 for C-H stretching, 1274.02cm^-1 for C-O alcohol stretching and 1186.37cm^-1 for C-O ketone were exhibited by AI and also CO exhibited a characteristics band at 2921.95 cm^-1 indicating the presence of (C-H stretching group), 2852.67cm^-1 (C-H stretching group). The 1709.84cm^-1 for carbonyl groups (C=O), 1492.29cm^-1 for CH₂ bending, 1379.21cm^-1 for NO₂ nitro compound,1269.96cm^-1 for C-C stretching,1186.02cm^-1 C-N amines,1081.11cm^-1C-O alcohols and 969.40cm^-1 for vinyl (C=C bending) were exhibited by CO. Significant changes have been recorded in the functional groups by observing the position and relative intensities of the band in IR.

 

HPTLC Fringier Printing:

The HPTLC fingerprinting of the chloroform extract of DP leaves, AI stem and CO whole plant are shown in Fig. 4A, 4B and 4C respectively. The results showing number of peaks, maximum R_f value, maximum height and total % area are given in Table4. The figure 4A, 4B and 4C clearly indicate that all samples constituents are clearly separated without any tailed and diffuseness. It is evident from Fig 4 that the chloroform extract CO shows more number of peaks (7 peaks) as compared to DP (5 peaks) and AI (4 peaks).

 

 

Figure 4: HPTLC chloroform extract of (A) Diplocyclos palmatus (DP), (B) Abutilon indicum (AI) and (C) Cassia occidentalis (CO)

 

The CO shows 7 peaks and the maximum percentage area covered is by peak 2 (Rf value 0.92 and 0.95) (Fig.4C). The DP shows 5 peaks and the maximum percentage area covered is by peak 1(Rf value 0.00) (Fig. 4A). The AI shows 4peaks and the maximum percentage area covered is by peak 1 (Rf value 0.96) (Fig 4B). The HPTLC results clearly reveals that the more number of phytochemical are present predominantly in CO extract as compared to DP and AI. The other components present in the extracts are found to be less predominant and found in less concentration.

Table 4:  HPTLC of Chloroform for DP leaves, AI stem and CO whole plant

 

The proposed HPTLC methods gives well resolved peaks for chloroform extract of CO, DP and AI. Based on result obtained. It is concluded that the method is sensitive, accurate, precise and less time consuming, it is very clear that the chloroform extract of CO, DP and AI containing not only a single compound but a mixture of compounds, this technique may be useful for identification and quality evaluation of plant. The chromatographic data and the information thus generated may be explored further as a tool for standardization.

 

GCMS Analysis:

The GCMS is most commonly used technique for the identification and quantification purpose. The unknown organic compounds in a complex mixture can be determined by interpretation and also by matching the spectra with reference spectra³⁹ thus, GCMS analysis is the first step towards understanding the nature of active principles of the medicinal plant and this type of study will be helpful for further detailed study. In the present study we analyzed the chloroform extract of DP, AI and CO using GC-MS in order to identify the number and type of phytochemicals present in them in figure 5. The components present in chloroform extract of CO, DP and AI were identified by GC-MS analysis (Fig 5). The name of phytochemicals with their retention time (RT), % peak area, molecular formula, molecular weight and chemical structure are shown in Table 5

 

 

Figure 5: GCMS chloroform extract of (A) Diplocyclos palmatus (DP), (B) Abutilon indicum (AI) and (C) Cassia occidentalis (CO)

 

The result clearly reveals the presence of total of 18, 16 and 12 compounds in DP, AI and CO respectively. The major component present in DP is 1-pentadecanol (24.41%) and the other components of DP includes 2(4H) benzofuronone (15.16%), 1-nonadecne (12.60%), benzene acetic acid (11.11%), dodecanoic acid (6.10%), pyrolidine 5-one (4.42%), propanedioic acid (4.01%), 4-piperidinepropanoic acid (3.05%) and L-proline-5 oxo-methylester (0.78%). The major component present in AI is 1-Heneicosylformate (18.60%) was found as major components followed by AI-heptadecane (8.04%),1,2,benzenedicarboxalic acid (7.39%),1-pentadecanol (7.20%), hexadecanoic acid (6.20%), phenol 2,4 bis(1,1dimethyl-ethyl)phenol(5.20%), hexadecane (4.90%), 9-octadeconoic acid(4.14%), 1-octadecene(3.96%), 1-nonadecene(3.39%), heneicoane(3.23%), 1-octadecane(2.39%) and docosane(1.62%).

 

Table 5: GCMS of Chloroform for DP leaves, AI stem and CO whole plant

Sr. No	Compound name	M.W	R.T	% Area	Structural formula
		(A) Diplocyclos palmatus
1	Phenol Formula: C6H6O	94	8.03	7.92
2	Phosphonic acid(p-hydroxyphenyl) phosphonic acidC6H7O4	17	11.02	1.88
3	Benzoic acid:C7H6O2	122	11.32	3.01
4	4-Piperidinepropanoic acid:  C19H26CINO3	351	12.31	3.05
5	Pyrrolidine-5-one:C7H13NO2	143	16.03	4.42
6	2-Pyrolidine:C5H9NO2	115	16.48	0.87
7	L-Proline,5-oxo,methyl ester proline: C6H9NO3	143	16.69	0.78
8	3-Oxo-4-phenylbutyronitrile:C10H9NO	159	16.91	1.60
9	Propanedioic acid:C9H8O4	180	16.99	4.01
10	Benzeneacetic acid: C8H8O2	136	18.27	11.11
11	3,5-bis(1,1-dimethyl) phenol:C14H22O	206	19.20	3.07
12	Dodecanoic acid: C12H24O2	205	19.62	6.10
13	2(4H)-Benzofuranone: C11H16O2	180	19.73	15.16
14	1-Pentadecanol: C15H32O	228	20.97	24.41
15	1-Nonadecne: C19H38	266	26.52	12.60
16	Di-n-octyl phthalate: C24H38O4	390

 

		B) Abutilon indicum
1	Phenol, 2, 4 bis (1,1dimethylethyl) Phenol: C14H22O	206	16.01	5.20
2	1-Nonadecene: C19H38	266	16.90	3.39
3	1-Octadecene:C18H36	252	16.92	3.96
4	1-Pentadecanol: C15H32O	228	19.19	7.20
5	Heptadecane: C17H36	240	19.25	8.04
6	Octadecane: C18H38	254	20.17	4.06
7	Heneicosane: C21H44	296	20.81	3.23
8	1-Octadecane: C18H38O	270	20.93	2.39
9	Hexadecane: C16H34	226	20.89	4.90
10	Docosane: C22H46	310	21.13	1.62
11	Hexadecanoic acid: C17H34O2	270	23.12	6.20
12	1, 2-Benzenedicarboxylic acid: C16H22O4	278	21.25	7.39
13	Phthalic acid butyl ester: C18H24O6	336	21.30	7.35
14	9-Octadecanoic acid C21H34O2	354	23.16	4.14
15	Naphthalene:C16H12	204	-
16	Pyrene:C16H10	202	-
17	1-Heneicosyl format: C22H44O2	340	26.51	18.60
18	Bacteriochlorophyll-c- stearyl: C52H72	840	-

 

		(C) Cassia occidentalis
1	4-Hydroxy-3-methyl acetophenone: C9H10O2	150	13.48	1.37
2	Phenol, 2,4-bis(1,1dimethyl) phenol: C14H22O	206	16.03	6.24
3	Hexadecane: C17H36	240	17.00	4.08
4	Octadecane:C18H38	254	17.35	4.62
5	Docosane: C22H46	310	19.27	9.48
6	Asarone: C12H16O3	208	20.61	0.13
7	Heneicosane: C21H44	296	20.99	21.09
8	Hexadecanoic acid: C17H34O2	270	26.53	6.34
9	Octadecanoic acid: C21H38O4	354	26.82	7.27
10	1, 2-Benzenedicarboxylic acid: C24H38O4	390	27.92	9.39
11	Nonacosane: C29H60	408	-
12	Tetratetracontane: C44H90	618

 

The major component present in CO is CE is heneicosane (21.09%) and the other components of CO includes docosane (9.48%), 1, 2 benzenedicarboxylic acid (9.39 %), octadeconoic acid (7.27%), hexadeconoic acid (6.34%). The peak indicates that the abundance of a constituent was different in plant. The position of peaks shows the time of elution which is different for each constituent because of its different structures. The heights of the peak indicate the relative concentrations of the components present in the plant. The mass spectrometer analyzes the compounds eluted at different times to identify the nature and structure of the compounds

 

Accelerated Stability Testing:

Suspensions were subjected for accelerated stability studies ie organoleptic characteristics such as the odour and colour of formulation were evaluated after 1 hr of preparation at room temperature. The accelerated stability studies at 8°c, room temperature and at 45° c were aimed to verifying the stability of the formulations developed for chemical, biological and physical stability. The results are expressed. In organoleptic studies, suspension was light green to olive green and suspension had a pleasant appearance and acceptable odour after 1h of preparation. On ageing, it was observed that suspensions did not exhibit any change in colour. The pH values of formulation after 1 hr were 6.49, 6.45, 6.4, 6.38, 6.58, 6.59, 6.5, 6.59, and 6.59 respectively. After ageing the values remained more or less constant and there is no chemical change. The sedimentation volumes of formulation were particle flocculated no caking was observed during this period and showed good redispersibility. The redispersibity was relatively constant, indicating that the suspension was easily redispersibity to yield uniform suspension even after storage for 90 days at 8°C and at 45°C (supplemental table no 6).

 

Table 6:  Accelerated Stability testing of formulation

Sr. No	Parameters	Initial	I month
			RT	8°C	45°C
01	Nature	Suspension	Suspension	Suspension	Suspension
02	Odour	Characteristics	Characteristics	Characteristics	Characteristics
03	Colour	Olive Green	Olive Green	Olive Green	Olive Green
04	Texture	Suspension	Suspension	Suspension	Suspension
05	Sedimentation  rate and volume	Particle Flocculated	Particle Flocculated	Particle Flocculated	Particle Flocculated
06	Rediapersibility	Good	Good	Good	Good
07	PH (10%w/v)	6.49	6.45	6.4	6.38

 

Table 6 Continue……

Sr. No	II Month			III Month
	RT	8°C	45°C	RT	8°C	45°C
01	Suspension	Suspension	Suspension	Suspension	Suspension	Suspension
02	Characteristics	Characteristics	Characteristics	Characteristics	Characteristics	Characteristics
03	Olive Green	Olive Green	Olive Green	Olive Green	Olive Green	Olive Green
04	Suspension	Suspension	Suspension	Suspension	Suspension	Suspension
05	Particle Flocculated	Particle Flocculated	Particle Flocculated	Particle Flocculated	Particle Flocculated	Particle Flocculated
06	Good	Good	Good	Good	Good	Good
07	6.58	6.59	6.5	6.59	6.59	6.59

 

Anticonvulsant Activity:

Epilepsy is characterized by recurrent episodes of seizures. A seizure is due to abnormal discharge of some neurons in the brain. Antiepileptic drugs may have a stabilizing influence on neuronal membrane; prevent detonation of normal brain cells by the focal discharge, these drugs act only on those neurons which are firing repeatedly. Some drugs reduce low threshold Ca++ current and abolish absence seizures whereas some drugs increase GABA activity in the synapse causing neuronal inhibition hence antiseizure effect. The ability of compound to prevent MES is believed to correlate with its ability to prevent spread of seizure discharge through neural tissue. Whereas the ability of compound to prevent threshold seizures (PTZ), has been correlated with the ability to raise the threshold for excitation of neural tissue.  Inhibition of the MES test predicts activity against generalized tonic-clonic and cortical focal seizures, lack of activity against MES induced seizures suggests that the drugs are effective in suppressing seizures. ⁷⁴ Herbal formulations were screened for anticonvulsant activity using MES induced convulsion model in rat and to produced anticonvulsant effect as compare to standard (Phenytoin 25mg/kg i.p.). Formulation exhibited flexion 11.83±1.47 seconds, hind limb extension for 38.16±3.65, clonus 139±2.36 and stupor 255.66±6.62 seconds respectively while compared with control group showed significant protect the animals from seizures and duration of each phase. It clearly indicates that formulation showed anticonvulsant activity expressed in table7 and fig 6A. After PTZ administration animal treated standard PTZ and diazepam showed clonic convulsion 93.33±3.55 seconds and formulation showed 55.83±5.49 seconds inhibits the seizures in table 8 and fig 6B.

 

Table 7: The Effect of MES Induced convulsions of Herbal formulation

Drug	Dose mg / kg b.w	Route of  Administration	Time (Sec) in various phases of convulsions (Mean + SEM				Recovery/ Death
			Flexion	Extensor	Clonus	Stupor
Control (Salaine)		Oral	41.83±1.94	53± 2.19	157.66±4.50	334.16±4.53	Recovery
Standard Phenytoin	25	Intra peritoneal(i.p )	10.5±1.51**	28.83±3.25**	119.16±2.63**	211.5±3.01**	Recovery
Formulation	200	Oral	11.83±1.47*	38.16±3.65*	139±2.36*	255.66±6.62*	Recovery

Data analysed using one way ANOVA, values are Mean± SEM, N=6 ***p<0.05, compared with std group

 

 

Table 8: The Effect of PTZ Induced convulsions of Herbal formulation

Drug	Dose  mg / kg b.w	Route of Administration	Onset of  convulsions (Sec. ± SEM)			Mortality %
			Onset	No. of Animal Survived	No. of Animal Convulsed
Control PTZ	80	Intra peritoneal (i.p)	36.67± 2.94	0/6	6/6	100.0%
Standard Diazepam +PTZ	4+80	Intra peritoneal (i.p )	93.33± 3.55	6/6	0/6	0%
Formulation	200	Oral	55.83± 5.49	6/6	4/6	0%

Data analyzed using one way ANOVA, values are Mean± SEM, N=6 ***p<0.05, compared with std group

 

Figure 6: Graph of herbal formulation for (A) MES and (B) PTZ induced convulsion

 

 

 

CONCLUSION:

In conclusion, extracts of DP leaves, AI stem and CO whole plant possesses anticonvulsant effects. The results of this experimental animal study lend pharmacological credence to the folkloric and ethno medicinal uses of the plant in the management of convulsive disorder. Oral herbal dosage forms of DP leaves, AI stem and CO whole plant in combination showed good palatability and having good stability on storage, could be formulated as herbal oral suspensions with convincing quality control parameters. However, this claim demands further study on individual formulations observing their comparative effect. Also the study needs elaborate technique to develop other suitable formulations from these extracts.

 

ACKNOWLEDGEMENT:

Authors are thankful to Mr. G. D. Patil, Secretary, Shree Warana Vibhag Shikshan Mandal, and Principal, Tatyasaheb Kore College of Pharmacy, Warananagar for their continuous support in carrying out this project.

 

CONFLICT OF INTEREST:

The authors declare that there are no conflicts of interest and no funding, self financed

 

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