INTRODUCTION:

World Health Organization recommends that various traditional medicine of treatment followed in different countries should be investigated. Many of the traditional medical practice medicines are not well established in scientific parameters^1,2. Discovery of bio-active constituents from the medicinal plants plays a key step in the modernization of medical World. Hence, the present study was aimed to evaluate the acute toxicity study and in vivo cardio protective activity of the ethanolic extract of Barleria cristata Linn. using Daphnia magna as a model^3,4.

Barleria cristata Linn is a shrub found widely in subtropical Himalayas, Sikkim, Khasi hills, Central and Southern India at a height of 1,350m.

It can also be found growing along roadside, slopes, streams and xeric vegetation at elevations below 100 m to 2600 m and as semi-natural habitats in dry & wet regions. It also been grown as ornamental plant in gardens^5,6,7.

Scientific Classification^8.

Domain : Eukaryote

Kingdom : Plantae

Phylum : Spermatophyte

Subphylum : Angiospermae

Class : Dicotyledonae

Order : Scrophulariales

Family : Acanthaceae

Genus : Barleria

Species : cristata

MATERIAL AND METHODS:

Pharmacognostical studies^9:

Collection, identification and authentication:

The fresh leaves of Barleria cristata Linn. was collected from Cuddalore district, Tamil Nadu, India in the month of March 2019. The plant material was identified and authenticated by Research Officer Dr K. N. Sunil Kumar, Research Officer and HOD Pharmacognosy, Siddha Central Research Institute, Arumbakam, Chennai. The authentication certificate number is 128.12031901-03.

Macroscopy:

Macroscopical study of the plant includes morphological features and organoleptic characters of the plant. Morphological characters like shape, size, surface, fracture and thickness were observed by naked eye or by magnifying lens or by dissecting microscope. Organoleptic characters like colour, odour, taste and nature were studied for the determination of identity, safety, efficacy and purity of crude drugs.

Microscopy:

Microscopical characters of the plant includes the anatomical study of the plant which is done by taking thin sections of the fresh leaves of the plants. Anatomical study is the most important distinguishing characters of the plant to prove their identity. Each distinguishing character are observed and presented¹⁰.

Quantitative microscopy – Linear measurement:^11-15

Quantitative microscopy was done as per the procedure given by Wallis (1962), Trease (1961) and Trease and Evans (1978). Measurement of cell constants of the crude drugs helps in their identification, characterization and standardization. So, the measurements of starch grains and trichomes were performed.

Determination of Leaf constants^16,17

The leaf constants like stomatal number, stomatal index, vein islet number and veinlet termination number were determined for B. cristata Linn. as per the standard procedure of Trease and Evans (1978).

Physicochemical constants^18-21

Foreign organic matter:

The determination of foreign organic matter was done as per the standard procedure given in Indian pharmacopoeia.

Ash values:

Total ash, acid insoluble ash, sulphated ash and water-soluble ash were determined as described in British Pharmacopoeia.

Extractive values:

Successive extraction with organic solvents in the order of increasing polarity was carried out as described in British Pharmacopoeia.

Loss on drying:

A quantity of 1g of powdered drug was taken in a glass stoppered weighing bottle and dried in a hot air oven at 105⁰C until to get a constant weight, cooled and weighed.

Foaming index:

The determination of foaming index can be done as per the procedure given by WHO.

Foaming index was calculated using the following formula

Foaming index = 1000/A

Where, A = Volume in ml of decoction used for preparing the dilution in the tube where foaming index was observed.

Florescence analysis:

The florescence character of powdered leaves and the extracts of Barleria cristata Linn. was studied both in day light and UV light and the results were recorded²².

Determination of heavy metals:

Heavy metals such as lead, copper, mercury, cadmium, arsenic are extremely toxic in very small amounts. The metals chosen for this study were arsenic and lead since they are highly toxic in very small amounts and the limit test for the above was performed as per Indian pharmacopoeia^23-25.

Preparation of extract:

The freshly collected leaves were washed with water, shade dried and coarsely powdered. The powdered material was defatted using pet. ether by maceration method for 7 days. Then filtered and dried. To the dried powder, ethanol is added and again kept for maceration for about 7 days. It is then filtered and evaporated by using rotary evaporator. The extract obtained is weighed and percentage is calculated in terms of air-dried weight of plant material. The colour and consistency of the extracts are noted and preserved in a refrigerator for further evaluation

Phytochemical studies:

Phytochemical evaluation is used to evaluate the nature of phytoconstituents present in the plant by using suitable chemical tests. Preliminary phytochemical analysis was carried out on the ethanol extract of Barleria cristata^26-33.

Culture of Daphnia magna:

D. magna was obtained from the local aquarium in Chennai, Tamil Nadu, India. It was identified and authenticated by Dr. P. C. Sathya Narayanan. D. magna were cultured by using Elendt-Bias (M4) and photoperiod + 12 hr were maintained. Spirulina used as a feed in spring water aerated for 48 hr to obtain oxygen concentration not less than 4 mg/ml. Experiment were carried out at 20^oC + 2^oC.

Experimental set-up:

The main component was a light microscope (Model), a digital video camera (PANASONIC, Model GP-KR222) Nikon cool pix camera (S9300) and an assembled computer. Image information was displayed on a monitor and recorded.

Fig 1. Daphnia magna

Assessment of Acute toxicity of Barleria cristata leaves on D. magna:

For the acute toxicity study, neonates with less than 24 hrs old were selected, since neonates may be more susceptible to toxic substance than the elder ones. The neonates were more specificity, simple and easy to handle in the lab. No feed was given throughout the study. Temperature is maintained at 20^oC+2^oC. The different concentrations of 1, 2, 3, 4, 5 and 6µl/ml extracts of the leaves of Barleria cristata was added directly to the 100 ml of spring water. D. magna is exposed to the extracts in six different concentrations for 48 hrs. Mortality rate was observed after 24 hr and LD50 was determined^34-37.

Cardiac arrhythmia:

Heart rhythm problems occur when the electrical impulses that coordinates the heart beats which does not work properly, causing the heart to beat too fast or too slower irregularly. Treatment of cardiac arrhythmia can often control or eliminate fast, slow or irregular heartbeats. In addition, cardiac arrhythmia is often made worse or even caused by a weak or damaged heart and it may be able to reduce arrhythmia risk by adopting a heart-lifestyle complications may include stroke and heart failure³⁸.

Medical procedure for treatment:

Cardio-version: Using electrical shocks to restore normal heartbeat in people with irregular heartbeats.

Radio-frequency ablation: Removal of tissue using the heat from an electrical current produced by a radio wave^.

In vivo Cardio protective activity:

D. magna of 10 days old were used. D. magna were separately placed in beakers containing 50ml of drug solution in different concentrations of 20, 40, 60, 80 and 100µg/ml of the test drug, control and standard metoprolol. D. magna were maintained at 20⁰C+2⁰C temperature during the experiment³⁹.

Recording of heart rate:

D. magna were transferred to the depression slide slightly coated with petroleum jelly. If most of the water is withdrawn, the animals are maintained in place by the surface tension of the remaining fluid and unable to displace out of the microscope’s field. The heart rate of control, standard and test drug treated groups were observed under light microscope³⁹.

Image processing:

The image processor allowed real time operations (i.e., 25 frames/s with a PAL or CCIR video camera as the input source), which was essential for analyzing fast heart movements. An electronic circuit on the card ensured jitter-free image captures, also from video recorders. This allowed the acquisition of already-processed and recorded data replayed on a video recorder, thus starting a further processing step. In this way, the implementation of complex algorithms is possible, if the operation was divided into sequentially executable parts.

Statistics:

The data were analyzed by one-way ANOVA followed by the Tukey’s test.

RESULTS AND DISCUSSION:

Pharmacognostical Studies:

Macroscopy:

Barleria cristata Linn is a shrub having simple leaf, green in colour and bitter taste. Leaves are ovate in shape with entire margin and acute acuminate apex. The upper surface is glabrous and lower is pubescent, pinnate venation, opposite phyllotaxy, decurrent petiole and slight hairy texture.

Fig 2.1, 2.2, 2.3 Macroscopy of Barleria cristata

Microscopy:

The transverse section of leaf (Fig 3.1) shows typical dorsiventral structure. The leaf has prominent midrib and smooth, even and glabrous lamina. The epidermis of both the surface is single layered. Cells are rectangular and covered externally with cuticle. Diacytic stomata (Fig 3.2) are present in both upper and lower epidermis. Each stoma is surrounded by varying number of subsidiary cells. Covering trichomes (Fig 3.3) are present on the epidermis. Trichomes occur on abaxial side of leaf, petiole and stem.

Fig 3.1 Fig 3.2

Fig 3.3

Fig 3.1, 3.2, 3.3 Microscopy of Barleria cristata

Powder microscopy:

Fragments of epidermal cells with diacytic stomata, covering trichomes and colourless oval shaped starch grains were seen.

Fig 4 Starch grains

Quantitative microscopy -Linear Measurements:

The results of length and width of starch grains and trichomes were measured and tabulated.

Table 1. Linear measurement of Starch grains of Barleria cristata

Parameters	Minimum (µm)	Average (µm)	Maximum (µm)
Length	14	31.5	56
Width	14	20.3	28

Table 2. Linear measurement of Trichomes of Barleria cristata

Parameters	Minimum (µm)	Average (µm)	Maximum (µm)
Length	126	378	630
Width	28	49	70

Determination of Leaf constants:

The results of leaf constants like stomatal number, stomatal index, vein islet number and veinlet termination number. Linear measurement of trichomes and starch grains were reported as follows.

Table 3. Determination of Leaf constants of Barleria cristata

S. No	Leaf Constants	Values per sq.mm
1.	Stomatal Number	17 – 19
2.	Stomatal Index	55 – 65
3.	Vein Islet Number	8-10
4.	Veinlet Termination Number	12.4-15.6

Physicochemical constants:

Foreign organic matter:

The percentage of foreign organic matter in the given crude drug was found to be 1%w/w.

Determination of ash value:

The ash values of Barleria cristata are as follows.

Table 4. Determination of ash value of Barleria cristata

S. No	Parameters	Percentage w/w
1.	Total ash	15.13 %
2.	Acid insoluble ash	8.03 %
3.	Water soluble ash	4.07 %
4.	Sulphated ash	10.23 %

Determination of extractive value:

The extractive values of Barleria cristata Linn. are as follows.

Table 5. Determination of extractive value of Barleria cristata

S. No	Parameters	Percentage w/w
1.	Water soluble extractive value	12 %
2.	Alcohol soluble extractive value	18.2 %

Loss on drying:

The loss on drying of the drug was found to be 1.4% w/w.

Foaming index:

Foaming index value was found to be less than 100.

Fluorescence analysis:

The Fluorescence analysis of leaf powder of Barleria cristata. are as follows^.

Table 6. Fluorescence analysis of leaf powder of Barleria cristata

S. No	Treatment	Day Light	Short UV Light (254nm)	Long UV Light (365nm)
1.	Powder	Dark green	Greenish black	Black
2.	Powder + water	Light green	Fluorescent green	Green
3.	Powder+1N Alc. NaOH	Light green	Fluorescent green	Greenish yellow
4.	Powder+1N Alc. KOH	Light green	Fluorescent green	Green
5.	Powder+1N H₂SO₄	Pale green	Light green	Pale green
6.	Powder+1N HCl	Light brown	Green	Brown
7.	Powder+1N HNO₃	Yellow	Fluorescent green	Dark green
8.	Powder+1N NaOH	Yellow	Fluorescent green	Green
9.	Powder+1N KOH	Light yellow	Fluorescent	Light green
10.	Powder+ acetic acid	Light green	Light brown	Purple
11.	Powder+ ammonia	Light green	Light green	Light green
12.	Powder+ ethanol	Green	Green	Yellowish brown
13.	Powder+Fecl₃	Brown	Fluorescent	Black
14.	Powder+ iodine	Yellowish brown	Light green	Brown

Determination of Heavy metals:

The determination of heavy metals like arsenic and lead were performed in the dried leaves of Barleria cristata. The results showed that the arsenic and lead was within the permissible limits, which was set by Food and Agricultural Organization (FAO) and World Health Organization (WHO) for human consumption.

Phytochemical Analysis:

Qualitative phytochemical analysis

The ethanol and aqueous extract, dried powdered leaves of Barleria cristata were subjected to preliminary phytochemical screening. The results of phytochemical studies are given in the following table.

Table 7. Determination of preliminary phytochemical screening of Barleria cristata

S. No	Plant constituent test/reagents used	Pet. ether	Ethanol	Aqueous	Powder
1.	Carbohydrates Molisch’s test	-	+	+	+
	Reducing sugars	-	+	+	+
	Gums & Resins	-	-	-	-
	Mucilage	-	-	-	+
2.	Proteins	-	+	+	+
3.	Amino acids	-	+	+	+
4.	Fats and oils	+	-	-	-
5.	Steroids	+	-	-	-
6.	Glycosides	-	+	+	+
	Cardiac glycosides	-	+	+	+
	Saponin glycosides	-	-	+	+
	Coumarin glycosides	-	-	-	-
	Flavonoids glycosides	-	+	+	+
7.	Alkaloids	-	-	-	-
8.	Phenols	-	+	+	+
9.	Tannins	-	+	+	+
10.	Terpenoids	-	+	+	+
11.	Quinones	-	+	+	+

‘+’ - denotes the presence of the respective constituent

‘-’ - denotes the presence of the respective constituent

Acute toxicity study:

The results of the acute toxicity study clearly reveals that no significant mortality was observed up to 6µg/ml of the ethanol extracts of the leaves of Barleria cristata Linn. The assessment of acute toxicity study reveals its safety and non-toxic nature.

Fig. 5 Acute toxicity study of the ethanol extract of Barleria cristata Linn.

Table 8. Mean Heart Beat (bpm)

Groups	Heart beat (bpm)
Control	172.6±2.24
Lactose (LACT)	86.5±1.99^a
Metoprolol (MET) + LACT	177.5±1.70^b
Test drug 20µg/ml + LACT	105±0.81^b
Test drug 40µg/ml + LACT	118.5±0.71^b
Test drug 60µg/ml + LACT	129.3±1.40^b
Test drug 80µg/ml + LACT	145.5±1.58^b
Test drug 100µg/ml + LACT	163.5±1.83^b

Values are expressed as mean± S.E.M, n=6 in each group,

^ap<0.001 LACT, when compared with Control. ^bp<0.001 MET + LACT,TD 1 + LACT, TD 2 + LACT,

TD 3 + LACT, TD 4 + LACT, TD 5 + LACT, when compared with LACT.

In vivo cardio protective activity:

The heartbeat of control, lactose, metoprolol and ethanol extracts of Barleria cristata Linn. namely 20, 40, 60, 80 and 100µg/ml treated were 172.6±2.24, 86.5±1.99, 177.5±1.70, 105±0.81, 118.5±0.71, 129.3±1.40, 145.5± 1.58 and 163.5±1.83 bpm respectively as shown in the bar diagram. The results revealed that the leaves of Barleria cristata Linn. has dose dependent cardio protective activity.

Fig 6. Heart rate of control and different concentrations of Barleria cristata Linn. treated on theLactose induced arrhythmic heart of Daphnia magna

CONCLUSION:

Pharmacognostical parameters help us to identify the plant and to carry out further studies on this plant. The overall results of this study provided promising baseline for the potential use of this plant Barleria cristata Linn. in the treatment of cardiac arrhythmia without any toxicity. From this study, this plant has been scientifically evaluated for treating cardiac arrhythmia. This scientific validation may be helpful for developing the herbal formulation using Barleria cristata Linn. to treat the cardiac arrhythmia. Further investigation on animal models and clinical trials are required to obtain drug leads.

ACKNOWLEDGEMENT:

We acknowledge our sincere thanks to Dr.K. Periyanayagam, Former Professor and Head, Department of Pharmacognosy, Madurai Medical College, who have been instrumental for this research work.

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Received on 04.09.2021 Modified on 21.03.2022

Research J. Pharm. and Tech 2023; 16(4):1587-1592.

DOI: 10.52711/0974-360X.2023.00259