Pharmacognostical Standardization and Pharmacological ... · 4162 Int J Pharm Sci Nanotech Vol 11; Issue 4 • July− August 2018 Research Paper Pharmacognostical Standardization

4162 Int J Pharm Sci Nanotech Vol 11; Issue 4 • July− August 2018

Research Paper

Pharmacognostical Standardization and Pharmacological Potential of Elaeocarpus ganitrus Leaves as an Antiulcer Agent

Mayank Kulshreshtha* and Manjul Pratap Singh School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh, India.

Received March 18, 2018; accepted May 9, 2018

ABSTRACT Elaeocarpus ganitrus Roxb, (E. ganitrus) known as Rudraksha belongs to family- Eleocarpaceae. It has a reflecting position in Hinduism and Ayurveda whereas traditionally it has mentioned to cure various health problems like fever, skin diseases, mental problems, wound healing etc. The present study was designed to study the microscopic and macroscopic analysis, physiochemical parameters, quantitative microscopy, phytochemical screening of E. ganitrus leaves as per WHO guidelines and evaluate the antiulcer potential of aqueous extract of E. ganitrus (AEEG) and ethanolic extract of E. ganitrus (EEEG) at the doses of 200 mg/kg and 400 mg/kg using pylorus ligation induced ulcers model, biochemical parameters. Hepatic, cardiac, hematological parameters have also done to find out the effect of different extracts on other major organs. Microscopic analysis proved the presence of covering trichomes, upper epidermis, lower epidermis, stomata, phloem, xylem etc. Ash value, water soluble ash, acid soluble ash, water soluble extract, alcohol soluble extract, loss on drying, swelling index, foaming index found to be 4.3 ± 0.52, 0.2 ± 0.33, 2.0 ± 0.2, 13.7 ± 0.25, 12.5 ± 0.55, 9.8 ± 0.23, 3.6 ± 0.04, more than 100. Different quantitative parameters were found out.

Phytochemical analysis of different extracts showed the presence of various primary and secondary metabolite like alkaloids, glycosides, tannin, phenolic compounds etc. Pharmacological potential showed that extracts treated, and sucralfate treated groups showed significantly decreases in ulcer index in all above-mentioned models, biochemical studies clearly showed significant decreases in volume, pH, free acidity, total acidity of gastric content and increases in gastric mucus parameters like protein, total hexoses, hexosamine, fucose, sialic acid and DNA level. The level of antioxidant enzymes like LPO (Lipid peroxidation), SOD (Superoxide dimutase) were decreased and CAT (Catalase) level was increased. Level of PC (Plasma corticosterone) was decreased. Hematological, hepatic, cardiac parameters found to be normal during extracts treatment. Histopathological analysis clearly supports the biochemical studies at various doses and it was found to be effective in dose dependent manner. The obtained scientific data may be helpful to prepare the monograph of the plant and E. ganitrus has antiulcer potential in a dose dependent. Detailed study needed for better exposure of plant.

KEYWORDS: Elaeocarpus ganitrus, pharmacognosy, antiulcer potential, histopathophysiology.

Introduction Peptic ulcer (PU) is a problematic issue may be acute

or chronic which is basically characterized by an imbalance between the lots of factors (aggressive & defensive) that harmful for the digestive tract (Nieto, 2012). Approximately 500,000 persons (78% between the ages of 25 to 64) develop PU in the United States each year (Sonnenberg et al., 1996). It is a GIT disorder that particularly affects the area that is more prone to acid damage (Helms et al., 2006). Some other researchers suggest that vagal control may be one of the reasons for the disturbances of gastric juice (Avram, 1976). Secretion of gastric acid is a typical process in which multiple factors play an important role like acetylcholine, histamine, gastrin, prostaglandins, these specific factors present on the basolateral membrane of the stomach (Romanes et al., 1986). Elaeocarpus ganitrus Roxb. (E. ganitrus), an evergreen tree contains Rudraksha, hard and highly ornamental

stony structure known as bead (Asolkar et al., 1992). Ayurveda refers to these strengthening body constitutions and may be worn either on wrist, arm or other parts of the body (Chopra et al., 1956). It is used in stress, anxiety, depression, palpitation, nerve pain, epilepsy, migraine, lack of concentration, asthma, hypertension, arthritis and liver diseases. According to the ayurvedic medicinal system, wearing of Rudraksha can have a positive effect on heart and nerves (Sakat et al., 2009: Singh et al., 2013: Khare, 2004: Gupta et al., 1984). The various scientific reports are available on various pharmacological activities include anti-inflammatory (Nain et al., 2011), antihypertensive (Sakat et al., 2009), antidepressant (Bhattacharya et al., 1975), central analgesic (Almeida et al., 2004), antioxidant (Kumar et al., 2008), antimicrobial (Singh et al., 1998), antimalarial and cytotoxic (Pouplin et al., 2007), antidiabetic (Bualee et al., 2007), and anti-asthma properties (Singh et al., 2000).

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 11 • Issue 4 • July – August 2018MS ID: IJPSN-3-18-18-KULSHRESHTHA

4162

Kulshreshtha and Singh: Pharmacognostical Standardization and Pharmacological Potential of Elaeocarpus ganitrus 4163

Our literature survey as from all possible sources revealed that there is little scientific data available for the pharmacognostical parameters and antiulcer potential of E. ganitrus leaves extracts at different doses. The present study was designed to study the microscopic and macroscopic analysis, physiochemical parameters, quantitave microscopy, phytochemical screening of E. ganitrus leaves as per WHO guidelines and evaluate the antiulcer potential of aqueous extract of E. ganitrus (AEEG) and ethanolic extract of E. ganitrus (EEEG) in experimental models of peptic ulcers.

Material and Methods Collection and Authentication of Leaf Material

The leaves of E. ganitrus were collected from National Botanical Research Institute, Lucknow and authenticated by Dr. Sunita Garg, NISCAIR, Delhi (Ref No. NISCAIR/RHMD/2015/2862/55-3).

Drugs and Chemicals

Methanols, Toluene, Formic acid, Ethyl acetate were purchased from SDFCL, Mumbai. Ethanol purchased from Changshu Yangyuan chemical, China. Sucralfate was provided as a free gift sample from Moraceae Pharmaceuticals Private Limited, Lucknow.

Extraction of Leaves Material

Leaves were shade dried and powdered by using electric grinder of 60 mesh size. Extraction was performed by soxhlation method. The powdered plant material was completely dried and defatted under Soxhlet assembly using 250 mL of petroleum ether for more than 6 hours after that the powder were dried and treated with different alcohols. The final alcoholic fraction obtained was concentrated under vacuum in a rotary evaporator at 40 °C and stored at 4°C for further use (Verma et al., 2014).

Animal’s Approval, Source and their Housing

Wistar rats weighing between 150-200g were used for the study after approval of Institutional Animal Ethics Committee (IAEC) under the Registration Number 809/PO/Re/S/03/CPCSEA by Ref No: BBDNIIT/IAEC/ 2017/05. Animals were purchased from Indian Veterinary Research Institute, Bareilly and kept in Babu Banarasi Das, Northern Indian Institute of Technology (BBDNIIT), Lucknow (U.P), India. The animal’s room conditions were maintained with natural light and dark cycle at controlled room temperature of 20-25 º C and they were left for 7 days for acclimatization to animal room on standard pellet diet and water ad libitum.

Pharmacognostic Analysis of Leaves Macroscopic & microscopic analysis: The macroscopic

features like color, size, shape etc. of the leaves were studied followed the method of Evans with the help of digital camera of Sony with 10.1 megapixel & for microscopic features of leaves were studied with the help of midrib. The very thin sections of leaves were treated

with 5% potassium hydroxide (KOH) and 20% chloral hydrate for removed of chlorophyll and fatty substances. Images were taken with image analyzer (Olympus Microscope with YOKO CCD Camera) (Evans et al., 2009).

Analysis of quantitative microscopy: The different parameters of fresh leaves like stomatal number, stomatal index, vein-islet number, vein termination number were performed according to WHO guideline (WHO, 1992).

Analysis of powder material of leaves: The completely air-dried plant material was first washed with distilled water, dried through air then powdered through mechanical grinder and stored in an airtight container for pharmacolognostical/ pharmacological analysis or development of the formulation in future.

Analysis of physical constituents of powder material: Physical constituents include loss on drying (Wallis, 2005) extractive values (Pharmacopoeia of India, 1996), ash value (Kokate et al., 2006: Ansari, 2006), swelling index (Joseph et al., 2011), foaming index (Mukherjee, 2007) were performed by different official methods.

Identification of Secondary metabolites by Phyto-chemical screening: Different secondary metabolites like carbohydrate, alkaloids, flavonoids, tannins, proteins, terpenoids etc. present in different leaves extracts of E. ganitrus (Solanki et al., 2011).

Pharmacological Screening for Antiulcer Activity Experimental groups: Lots of models are available for

the screening of newer compounds but in present investigation pylorus ligation (PL)- induced ulcers model have been chosen for exploring the antiulcer potential of various leaves extracts (Shay et al., 1945). Grouping of animals had done by the following way. Group 1 served as a negative control group which treated with inducers (Pylorus ligation), Group 2 served as a standard group which treated with Sucralfate (8.6 mg/kg), Group 3 served as test group 1 which treated with AEEG (200 mg/kg), Group 4 served as test group 2 which treated with AEEG (400 mg/kg), Group 5 served as test group 3 which treated with EEEG (200 mg/kg), Group 6 served as test group 4 which treated with EEEG (400 mg/kg). All extracts and standard drug were dissolved in 1% carboxy methyl cellulose and administered orally at 10.00 am and 4.00 pm respectively for 5 days for the antiulcer screening (Sairam et al., 2002).

Gastric secretion study: The different parameters like volume of gastric juice, pH, total acidity, free acidity have been done by different official methods. 1 mL of gastric juice collected in a conical flask and added some drops of Topfer’s reagent and titrated with 0.01 N sodium hydroxide until the red colour become disappeared and it became yellowish orange after that volume was noted and this referred to free acidity now 2 to 3 drops of phenolphthalein solution were added until the above color disappeared and it became red, volume was noted and refereed as a total acidity (Sener et al., 2004).

Analysis of gastric mucus: The glandular portions of stomach were removed and immediately homogenized


with some Ml of distilled water. The difference of the weight of water before and after homogenization yielded the weight of mucus. Aliquots were taken from the preparation for qualitative analysis of glycoprotein components like hexose, hexosamine, sialic acid, fucose, protein and DNA content were done by official methods (Winzler et al., 1958: Elson et al., 1933: Alaya et al., 1993: Lowrey et al., 1951: Goel et al., 1996).

In vivo Antioxidant Activity Lipid peroxides (LPO) activity: Malondialdehyde

(MAD) level was measured by thiobarbituric method in which the brains were homogenized in 50 mM phosphate buffer and centrifuged at 15,375 × g at 4 ºC for 20 minute after that deproteinised with 40% trichloro acetic acid (TCA) and 5M Hydrochloric acid, followed by addition of 2% (w/v) thiobarbituric acid in 0.5 M sodium hydroxide. The reaction mixture was heated in a water bath at 90 ºC for 15 minute and centrifuged at 12,000 × g for 10 minutes. The pink chromate formed was measured at 532 nm spectrophotometrically (Nichaus et al., 1986: Yaki, 1994).

Superoxide dismutase (SOD) activity: SOD was estimated by following the procedure of Kakkar et al., 1984. The inhibition of reduction of nitro blue tetrazolium to blue coloured formozan in presence of phenazine metha sulphate and NADH was measured at 560 nm using n-butanol as blank and has been expressed as U of SOD activity/mg protein (Kakkar et al., 1984).

Catalase estimation: A 10% homogenate brain was prepared with 1.95 mL ice-cold phosphate buffered Saline (0.05M, pH 7) mixed with 1mL hydrogen peroxide (0.019M) was centrifuged at 10,000 rpm at -4º C for 15 minute and the supernatant obtained was used for observation of changes in absorbance will be recorded at 240 nm. Results are expressed as U of CAT activity/mg protein (Nagakannan et al., 2012).

Estimation of Plasma Corticosterone

Plasma corticosterone was done by as the method of Glick et al., 1964.

Stomach Histopathology

Picric acid, Formaldehyde and 40% glacial acetic acid were used as fixative agents for washed stomach then the sample tissue dehydrated with alcohol, cleaned and fixed in paraffin. A very small piece (3-5μm) cut, stained with haemetoxylin and eosin and observed under microscope, evaluated the changes at cellular level (Mohamed et al., 2011).

Results and Discussion Morphology

E. ganitrus is compound, light green in color, acute apex, reticulate veins, 12 to 15 pairs of vein, slightly crenate to entire margin, oblong shape with symmetrical base. The size of leaf is average 15 cm long and 4cm in width. Leaf is bitter in taste and odorless (Fig. 1).

Fig. 1. Morphology of E. ganitrus leaf.

Microscopic Characters

Transverse section of E. ganitrus leaf (Midrib and Lamina) also showed the presence of the different areas such as upper epidermis, lower epidermis and mesophyll (Fig. 2). The upper epidermis and lower epidermis both were single layered covered with cuticle, polygonal, straight and few contain mucilage. The spongy parenchyma 5- 8 layered, tight, no intracellular spaces (Fig. 3). Covering trichomes were present on both of the epidermis and they were unicellular, long, dagger shaped with bulbose base. The middle portion showed the presence of xylem was lignified cells present at ventral surface where phloems were non-lignified present at dorsal surface (Fig. 4).

Fig. 2. Transverse section of E. ganitrus leaf.


Fig. 3. Transverse section of E. ganitrus leaf showing spongy parenchyma.

Fig. 4. Transverse section of E. ganitrus leaf showing Phloem and Xylem.

Powder microscopic evidence

Powder characteristic of E. ganitrus leaves revealed the presence of calcium oxalate crystals, epidermal cells, paracytic stomata, lignified trichomes, spring shape trichomes found in E. ganitrus as shown in Fig. 5.

Fig. 5. Powder characters of E. ganitrus.

Quantitative Parameters, Physicochemical Evidence and Phytochemical Screening

The quantitative parameters and physicochemical properties of the leaves were performed as per Ayurvedic Pharmacopoeia of India. Phytochemical testing of different extracts shows the presence of alkaloids, glycosides, sterols, carbohydrates, tannins, terpenoids,

saponin, flavonoids etc. as shown in Tables 1-3 respectively.

TABLE 1 Quantitative microscopy of leaf of E. ganitrus

Parameters E. ganitrus Vein islet number (1 mm² leaf surface) 64.35 ± 3.10 Vein termination number (1 mm² leaf surface) 64.48 ± 3.16 Stomatal number (1 mm² leaf surface on lower epidermis)

86.45 ± 1.67

Stomatal number (1 mm² leaf surface on upper epidermis)

134 ± 1.36

Stomatal index 6.20 ± 0.04 Palisade ratio 3.45 ± 0.03

TABLE 2 Physicochemical analysis of leaves of E. ganitrus

Parameters E. ganitrus Total ash 4.3 ± 0.52 Water soluble ash 0.2 ± 0.33 Acid insoluble ash 2.0 ± 0.02 Water extractive value 13.7 ± 0.25 Ethanol extractive value 12.5 ± 0.55 Loss on drying 9.8 ± 0.23 Swelling index 3.6 ± 0.04 Foaming index >100

TABLE 3

Phytochemical analysis of different extracts of E. ganitrus.

Phytoconstituents AEEG EEEG Alkaloid ++ + Glycosides + ++ Tannins + +++ Flavonoids + +++ Fats and oil + ++ Carbohydrates ++ +++ Reducing sugar + ++ Proteins +++ +++ Saponin + +++ Terpenoids ++ +++

AEEG- Aqueous extract of E. genitrus, EEEG- Ethanolic extract of E. genitrus.

Pharmacological Screening for Antiulcer Potential Effect of AEEG and EEEG on ulcer index in pylorus

ligation (PL) - induced ulcers: In case of pylorus ligation method, the minimum ulcer index found to be 3.2±0.44 at the dose of 400 mg/kg in case of EEEG which is a statically significant (p<0.001) whereas 3.9±0.46 at the dose of 200 mg/kg at the same case. In case of AEEG, the ulcer index found to be 4.5±0.52 and 4.1±0.49 at the 200 mg/kg and 400 mg/kg (Table 4).

TABLE 4

Effect of different leaf extracts of E. ganitrus on ulcer index in pylorus ligation (PL) - induced ulcers.

Group Treatment Pylorus ligation

Dose (mg/kg) Ulcer index (mm2/rat) 1 Negative control P.L 13.5 ± 0.94

2 Sucralfate 8.6 2.4 ± 0.45***

3 AEEG 200 4.5 ± 0.52** 4 AEEG 400 4.1 ± 0.49*** 5 EEEG 200 3.9 ± 0.46** 6 EEEG 400 3.2 ± 0.44***

Results were expressed as MEAN ±SEM, n=6 and *p<0.05, **p<0.001, **p<0.001, when compared with negative control analyzed by one-way ANOVA followed by Turkey test.


Effect of AEEG and EEEG on gastric juice in pylorus ligation (PL) - induced ulcers: Gastric juice analysis clearly indicated that the amount of gastric juice, free acidity, total acidity were decreased with the increases the dose in case of all three models where as pH was increased with increases the dose. All the data were statically significant at p<0.05, **p<0.001, ***p<0.001, when compared with negative control (Table 5).

Effect of AEEG and EEEG on gastric mucus in pylorus ligation (PL) - induced ulcers: From the Table 6, it is clearly justified that the herbal extracts are working in a dose dependent manner. The weight of mucus, protein, hexose, hexosamine, sialic acid, fucose, protein and DNA content were increased with increases the dose and statically significant (at p<0.05, **p<0.001, ***p<0.001).

Effect of AEEG and EEEG on hematological parameters, hepatic parameters, cardiac parameters in pylorus ligation (PL) - induced ulcers It is clearly observed in Table 7, 8 and 9 that there is no change of any type in hematological value, cardiac value and hepatic values in case of all models compare to all groups means the values that was found out during all three analysis did not changed for examples in case of

haematological parameters the values of HB, RBC, WBC, Urea, Sugar found to be 13.2 (g/dl), 6.6 (×106 /µL), 8.4 (×103 /µL), 17.02 (mg/dl), 96.15 (mg/dl) respectively in case of sucralfate (in PL induced ulcers) so these value are near about all treatment groups so it is proved that the test compounds did not show any side effect in blood, liver and heart apart from that interesting thing was found that the values of HDL were increased so it can be concluded the AEEG and EEEG also have the cardio protective property.

Effect of AEEG and EEEG on LPO (Lipid peroxi-dation), SOD (Superoxide dimutase), CAT (Catalase), PC (Plasma corticosterone) in pylorus ligation (PL) - induced ulcers: The values of LPO, SOD, CAT, PC were found to be 0.56±0.08 (nmol/g wet tissue), 149.4±2.26 (units/g wet tissue), 17.7±0.16 (units/g wet tissue) and 6.6±0.64 in case of AEEG at the dose of 200 mg/kg whereas 0.53±0.02 (nmol/g wet tissue), 148.7±2.22 (units/g wet tissue), 18.6±0.23 (units/g wet tissue) and 25.9±0.61 so it can be concluded the level of SOD, LPO and PC were decreased whereas CAT was increased with increase the dose of a test compounds. Theses all values are statically significant at p<0.05, **p<0.001, ***p<0.001 level (Table 10).

TABLE 5

Effect of AEEG and EEEG on gastric juice in pylorus ligation (PL) - induced ulcers.

Groups Treatments Dose

(mg/kg) Volume of

gastric content pH of gastric

content Free acidity

(mEq/I) Total acidity

(mEq/I)

1 Negative control P. L 2.24 ± 0.05 2.2 ± 0.04 58.33 ± 2.19 128 ± 4.25

2 Sucralfate 8.6 1.02 ± 0.01*** 6.2 ± 0.14*** 26.34 ± 0.21*** 26.2 ± 1.20***

3 AEEG 200 1.41 ± 0.08*** 3.6 ± 0.10*** 40.13 ± 1.05** 49.8 ± 1.23*

4 AEEG 400 1.36 ± 0.06*** 4.1 ± 0.12*** 39.12 ± 0.26*** 46.6 ± 1.19**

5 EEEG 200 1.45 ± 0.09*** 4.2 ± 0.13** 29.17 ± 0.24** 57.6 ± 2.01*

6 EEEG 400 1.39 ± 0.06*** 4.8 ± 0.11*** 22.14 ± 0.15*** 55.4 ± 1.19***

TABLE 6

Effect of AEEG and EEEG on gastric mucus in pylorus ligation (PL) - induced ulcers.

Groups Wt of mucus (gm) Protein (mg/g) Total Hexoses (mg/g)

Hexosamine (mg/g)

Fucose (mg/g) Sialic acid (mg/g) Total Carbohydrate DNA (µG/ml)

1 0.133 ± 0.001 36.67 ± 0.21 29.20 ± 0.10 35.32 ± 0.42 99.6 ± 1.64 1.58 ± 0.009 165.7 ± 1.29 93.6 ± 1.51* 2 0.182 ± 0.013*** 52.29 ± 1.16*** 42.29 ± 0.24** 73.28 ± 0.82* 139.3 ± 1.91** 4.24 ± 0.11** 259.11 ± 2.51** 246.5 ± 2.46* 3 0.156 ± 0.007*** 44.32 ± 0.32*** 39.29 ± 0.21* 62.21 ± 0.76** 132.5 ± 1.84*** 3.52 ± 0.09** 237.52 ± 2.36** 184.2 ± 1.65** 4 0.161 ± 0.008*** 51.67 ± 1.19*** 44.31 ± 0.27* 64.29 ± 0.74** 135.4 ± 1.84*** 3.61 ± 0.11** 241.67 ± 2.29** 188.3 ± 1.63* 5 0.162 ± 0.008*** 41.26 ± 0.26*** 40.16 ± 0.23*** 59.21 ± 0.73*** 124.5 ± 1.69** 3.51 ± 0.08*** 227.38 ± 2.19** 173.8 ± 1.54**

6 0.165 ± 0.011*** 44.08 ± 0.42*** 46.28 ± 0.31*** 61.23 ± 0.77** 131.3 ± 1.83** 3.54 ± 0.07*** 242.35 ± 2.31* 182.4 ± 1.58**

Results were expressed as MEAN ±SEM, n=6 and *p<0.05, **p<0.001, ***p<0.001, when compared with negative control analyzed by one-way ANOVA followed by Turkey test.

TABLE 7

Effect of AEEG and EEEG on hematological parameters in pylorus ligation (PL) - induced ulcers.

Groups Treatments Dose (mg/kg) HB (g/dl) RBC (×106 /µL) WBC (×103 /µL) Urea (mg/dl) Sugar (mg/dl)

1 Negative control P. L 14.5 ± 0.61 7.2 ± 0.89 8.9 ± 0.92 18.22 ± 0.98 115.14 ± 2.01

2 Sucralfate 8.6 13.2 ± 0.53*** 6.6 ± 0.23*** 8.4 ± 0.86*** 17.02 ± 0.69*** 96.15 ± 1.44**

3 AEEG 200 14.5 ± 0.65*** 6.9 ± 0.26*** 9.0 ± 0.96*** 17.09 ± 0.79*** 96.10 ± 1.43***

4 AEEG 400 14.4 ± 0.59*** 6.7 ± 0.24*** 8.6 ± 0.89*** 17.06 ± 0.74*** 96.12 ± 1.41*

5 EEEG 200 13.7 ± 0.56*** 6.8 ± 0.25*** 9.2 ± 0.97*** 17.12 ± 0.84*** 96.07 ± 1.41***

6 EEEG 400 13.6 ± 0.55*** 6.4 ± 0.23*** 8.4 ± 0.86*** 16.92 ± 0.61*** 96.09 ± 1.43***



TABLE 8

Effect of AEEG and EEEG on hepatic parameters in pylorus ligation (PL) - induced ulcers.

Groups Treatments Dose

(mg/kg) Total protein

(g/dl) Albumin

(g/dl) Globulin

(g/dl) ALP (U/L) ALT (U/L) AST (U/L)

1 Negative control P. L 7.09 ± 0.17 3.07 ± 0.46 4.02 ± 0.39 82.26 ± 2.46 14.06 ± 0.95 14.05 ± 0.95 2 Sucralfate 8.6 8.06 ± 0.19*** 3.22 ± 0.54*** 4.84 ± 0.45*** 78.02 ± 1.42*** 13.01 ± 0.93*** 10.32 ± 0.33*** 3 AEEG 200 8.16 ± 0.21** 3.16 ± 0.51** 5.00 ± 0.58* 76.48 ± 1.26** 12.28 ± 0.92** 10.29 ± 0.31** 4 AEEG 400 8.18 ± 0.20*** 3.19 ± 0.53*** 4.99 ± 0.50*** 77.12 ± 1.03*** 12.36 ± 0.93** 9.97 ± 0.26*** 5 EEEG 200 8.26 ± 0.23** 3.23 ± 0.54** 5.03 ± 0.59* 76.29 ± 1.24* 12.24 ± 0.91** 11.01 ± 0.35*** 6 EEEG 400 8.29 ± 0.24*** 3.13 ± 0.49*** 5.16 ± 0.54*** 78.23 ± 1.23** 11.26 ± 0.91*** 10.11 ± 0.29***


TABLE 9

Effect of AEEG and EEEG on cardiac parameters in pylorus ligation (PL) - induced ulcers.

Groups Treatments Dose (mg/kg) TC (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl)

1 Negative control P. L 64.32 ± 3.01 51.09 ± 1.98 18.19 ± 0.63 20.04 ± 0.99 10.02 ± 0.82 2 Sucralfate 8.6 53.31 ± 0.24*** 50.26 ± 1.73*** 20.19 ± 1.02*** 17.19 ± 0.74*** 9.53 ± 0.79*** 3 AEEG 200 51.14 ± 2.06** 49.15 ± 2.01** 20.32 ± 1.08** 18.51 ± 0.79** 9.97 ± 0.83*** 4 AEEG 400 50.12 ± 1.74*** 50.16 ± 1.72*** 21.29 ± 1.01*** 18.52 ± 0.80*** 10.02 ± 0.82*** 5 EEEG 200 52.13 ± 2.10*** 52.18 ± 2.15** 22.14 ± 1.12* 17.56 ± 0.83** 10.12 ± 0.79** 6 EEEG 400 50.16 ± 1.63*** 51.16 ± 2.08* 22.09 ± 1.08*** 18.01 ± 0.64** 10.11 ± 0.81***


TABLE 10

Effect of AEEG and EEEG on LPO (Lipid peroxidation), SOD (Superoxide dimutase), CAT (Catalase), PC (Plasma corticosterone) in pylorus ligation (PL) - induced ulcers.

Groups Treatments Dose (mg/kg) LPO

(nmol/g wet tissue) SOD

(units/g wet tissue) CAT

(units/g wet tissue) PC

1 Negative control PL 0.92 ± 0.04 216.5 ± 2.68 16.4 ± 0.12 36.2 ± 0.51 2 Sucralfate 8.6 0.42 ± 0.02*** 133.6 ± 2.11** 26.5 ± 0.23** 22.4 ± 0.34*** 3 AEEG 200 0.56 ± 0.08* 149.4 ± 2.26* 17.7 ± 0.16*** 26.6 ± 0.64** 4 AEEG 400 0.53 ± 0.02** 148.7 ± 2.22* 18.6 ± 0.23*** 25.9 ± 0.61*** 5 EEEG 200 0.57 ± 0.04** 148.3 ± 2.26** 17.8 ± 0.18*** 25.5 ± 0.58*** 6 EEEG 400 0.54 ± 0.03*** 145.7 ± 2.14** 18.8 ± 0.25*** 24.2 ± 0.52***


Fig. 6. Different sections of stomach treated with hematoxylin and eosin for histopathological analysis (A) Stomach of negative control group (PL) shows the deep ulcers loss of gastric mucosa, the ulcerative area are full of hemorrhage. Pyloric glands are totally ruptured; mucosa and sub mucosa are found to be very negligent stage. (B) Section of stomach treated with sucralfate shows the normal condition of all lawyers, glands and no ulcers. (C) Stomach section treated with AEEG (200 mg/kg) shows the lawyers are normal but some minute ulceration in gastric glands. (D) Stomach treated with AEEG (400 mg/kg) shows all parameters are normal but presence of small edema in section (E) Stomach section treated

with EEEG (200 mg/kg) shows low grade edema, very minute ulceration. (F) Stomach section treated with EEEG (400 mg/kg) shows only small edema.

Histopathological analysis of stomach

In PL method, negative control group shows the rupturing of all stomach lawyers, inhibition of pyloric gland and necrosis found out, the whole ulcerative area full of blood extra vasation (Fig. 6A). Appearance of sucralfate treated group is normal (Fig. 6B). AEEG (200 mg/kg) shows minute ulceration whereas appearance of AEEG (400 mg/kg) treated are normal (Fig. 6C and 6D). parameters of EEEG (200 mg/kg) shows minute edema and ulceration, layers are normal, but group treated with EEEG (400 mg/kg) shows normal parameters so histopathological analysis proved that extract has an ulcer protective activity in a dose dependent manner (Fig. 6E and 6F).

Conclusions

The present study clearly raveled those pharma-cognostic features of plant which are helpful to find out the anatomy, physiology, location and necessary in


formations regarding plant. AEEG and EEEG better antiulcer activity and it may be act as an antagonist of the chemicals which are responsible for the HCl secretion. Analysis of hematological, hepatic and cardiac parameters created the safety profile. It may be beneficial for the preparation of monograph of the plant, pharmacological research in near future or may be beneficial for the new researchers.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

I would like to express my hearty thanks National Botanical Research Institute (Lucknow) for the collection of plant leaves, Dr. Sunita Garg, NISCAIR, New Delhi for the authentication of leaves material, Dr. Amit, Scientist, Indian Veterinary Research Institute (Bareilly) for the procurement of animals, Dr. Gadiputi Shridhar (Prof & HOD), Department of oral pathology and microbiology, BBDCODS, Lucknow for histopathological studies and also thankful to Moraceae Pharmaceuticals Private Limited (Lucknow) for providing the sucralfate free gift sample.

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Address correspondefnce to: Mayank Kulshreshtha, Assistant Professor, Department of Pharmacology, School of Pharmacy, Babu Banarasi Das University, Lucknow, Uttar Pradesh, India Phone: +91-9897083663 E-mail: [email protected]

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Pharmacognostical Standardization and Pharmacological ... · 4162 Int J Pharm Sci Nanotech Vol 11; Issue 4 • July− August 2018 Research Paper Pharmacognostical Standardization