Results & Discussion

CHAPTER 3

RESULTS AND DISCUSSION

3.1 PLANT AUTHENTICATION

Leaves were selected on the base of traditional uses and extensive literature survey was done
to rule out similar work in the past. Literature survey was done from books, online sources
and Medicinal and Aromatic Plants Abstracts (MAPA) from 1979-2018.The Cyperus
rotundus Linn. were collected from Nagina , Dist. Bijnor, Uttar Pradesh. The drugs was
identified and authenticated by Mr. RS Jayasomu, The senior Principal Scientist, head of Raw
Material Herbarium and Museum (RHMD), CSIR NISCAIR Delhi India. Where a voucher
specimen (Voucher No-NISCAIR/RHMD/Consult/2018/4118-68-5) is maintained for further
reference.

3.2 MACROSCOPICAL CHARACTERS:

 Cyperus rotundus rhizomes are bluntly conical with number of wiry and tough slender roots,
frequently attached to each-other via thin and tough connective. Every rhizome is tumicate,
differ in size and weight, crowned with remains of trunk and leafs forming a hairy to scaly
covering.

Table 3.1 Observations of macroscopical characters

60
 Results & Discussion
3.3 CHEMICAL CONSTITUENTS PRESENT IN CYPERUS ROTUNDUS
RHIZOME:
The micro-chemical tests for rhizome powdered of Cyperus rotundus was performed and
cellulose, chitin, and fats, suberin, sudan red cutin, tannins, starch, mucilage, proteins,
calcium oxalate crystals, cellulose cell wall & calcium carbonate were present.

Table 3.2: Micro-chemical constituents of Cyperus rotundus rhizome

S.NO Constituents Test Observation Inference

1. Cellulose Iodine solution Pale yellow +

color

Iodine solution + Bright blue -

Sulphuric acid color

2. Chitin Iodine solution Deep blue color +

Iodine solution + Brown color +

Sulphuric acid

Safranin Red color +

Phloroglucinol + Phloem fibers +

conc. HCl stained red
color

3. Fixed oils and Chloroform Soluble +

fats

4. Suberin, Iodine solution Deep yellow +

Sudan red color
Cutin
Iodine solution + Deep brown +
Sulphuric acid color

61
 Results & Discussion
Iodine +Sudan red III Red color +
Solution

5. Tannins Dilute ferric chloride Blue black -

solution color

6. Starch Iodine solution Blue color +

7. Mucilage Methylene blue Deep blue color +

Iodine solution + Violet color +

Sulphuric acid

8. Proteins Iodine solution Yellow +

crystalloids

Alcoholic picric acid Yellow +

solution crystalloids

Million’s reagent Yellow color +

9. Calcium Powdered plant Dissolve +

oxalate material + acetic acid hydrochloric
crystals acid insoluble
acetic acid

10. Cellulose cell Iodine & sulphuric Blue violet +

wall acid solution colors

11. Calcium Acetic acid Dissolve with -

carbonate acetic acid

62
 Results & Discussion
3.4 PHYTOCHEMICAL CHARACTERS

3.4.1 Overall Ash content of Cyperus rotundus rhizome

The overall ash value of the plant was measure by subtracting overall wt of crucible and wt of
ash from weight of empty crucible. The overall ash value was found from Cyperus rotundus
which is 11 mg/g.

Table 3.3Overall ash value

Note: Results are the means of three observations of drug sample.

3.4.2 Acid-Insoluble Ash Value of Cyperus rotundus rhizome

The acid insoluble ash of the plant was measure by subtracting overall ash from wt of
crucible and weigh of acid insoluble ash. The acid insoluble ash value was found from
Cyperus rotundus rhizome which is 8.0 mg/g.

Table 3.4: Acid-insoluble ash

Note: Results were the means of three observations of drug sample.

63
 Results & Discussion
3.4.3 Water Soluble Ash Content of Cyperus rotundus rhizome:

The water soluble ash of the plant was measure by subtracting overall ash from wt of crucible
and wt of water soluble ash. Water soluble ash was found from Cyperus rotundus which is 4
mg/g.

Table 3.5 Water soluble ash

Note: Results were the means of three observations of drug sample.

3.5 PHYTOCHEMICAL SCREENING OF CYPERUS ROTUNDUS RHIZOME

EXTRACT

Phytochemical are non-nutritive, chemically active substances present in plants that create a
specific physiological action on the human body and perform an important function in the
handling of serious diseases. Since early times, the plant had been used in prevention and
treating of different diseases. In the current study the leaves residue of plants under study
were analyze for tannins, saponins, alkaloids, anthraquinone glycosides, protein & amino
acids ,lipids , carbohydrates, flavonoids and phenols as per standard protocols given below
[128,129]

64
 Results & Discussion

Table: 3.6 Obsevartion of phytochemical screening of extracts

S.NO Constituents CHEMICAL TEST Observation Inference

Hydro-
alcoholic
Extract

1. Alkaloids Mayer’s Test Yellow cream +

precipitates

Hager’s Test yellow +

precipitates
2. Anthraquinone Brontranger’s Test Pink -red colour +
Glycosides formed

Modified Brontrager’s Test Rose red colour +

formed

3. Protein Biuret Test Violet or pink +

formed
Xanthoproteic Test Yellow colour +
is obtained

4. Amino acid Ninhydrin Test Violet or purple +

color.is
developed
5. Carbohydrate Molisch’s Test Red or purple +
color formed

6. Tannins Dilute ferric chloride solution Blue black -

color

7. Saponins Foam Test Foam formation +

8. Flavonoids Shinoda Test Pink, crimson +

65
 Results & Discussion
or magenta
color is
developed
9. Phenols Ferric chloride Test Violet color is +
formed

10. Lipids Grease spot Test Oil layerd is +

formed

3.6 ORAL ACUTE TOXICITY

No death or perception of adverse reactions was observed on the animals within the fourteen
days follow up period. This is a signal that the extracts, were not toxic. From the acute oral
toxicity result it was clear that no symbol of mortality was found up to the dose of
2000mg/kg. Hence the 1/10th & 1/20th of this dose 200 and 100 respectively were selected.

3.7 Effects of the Hydro-alcoholic Extracts of Cyperus rotundus rhizome on

Heamodynamic parameters in Hypertensive rats:

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean blood pressure
(MBP) of ethanol-induced Hypertensive rats receiving purified water were significantly (p <
0.001) increases as comparing to normo-tensive rats (NTR). The administration of Cyperus
rotundus at the respective doses of 100mg/kg and 200mg/kg along with nifedipine (10mg/kg)
resulted in a significantly lowering (p < 0.001) of SBP, DBP, and MBP as comparing to
HTR. The extracts at the dose of 200mg/kg and nifedipine reducing the values of SBP, DBP,
and MBP to values close to those of normotensive rats. The plant extract at both doses (100
and 200 mg/kg) and nifedipine (10mg/kg) had no affect on the heart rate of hypertensive rats.

66
 Results & Discussion
TABLE 3.7 Effects of Hydroalcoholic extract of Cyperus rotundus on hemodynamic
parameters in Hypertensive rats:.

Each value represents the mean ± SEM (n = 5). BPM: beat per minute, SBP: systolic blood pressure, (DBP)
diastolic blood pressure, (MBP) mean blood pressure, HR: heart rate, and (NTR) normotensive rats receiving
distilled water (10 mL/kg), HRT: hypertensive rats receiving distilled water (10 mL/kg) and alcohol (5 g/kg),
C.R. 100 and C.R. 200: hyper-tensive rats receiving, ethanol and Cyperus rotundus extract at the doses of
100mg/kg and 200 mg/kg, respectively, and Nif. 10: HTR receiving alcohol and nifedipine at the dose of
10mg/kg; *: p <0.05 significant difference from normotensive rats.

67
 Results & Discussion
3.8 Effects of Hydro-alcoholic Extract of Cyperus rotundus on Lipid Parameters in
Hypertensive rats:

The affects of the Hydro-alcoholic extract of Cyperus rotundus on the serum level of overall
cholesterol, triglyceride, HDL-cholesterol and LDL-cholesterol. It shows that overall
cholesterol, triglyceride, LDL cholesterol significantly (p < 0.001) enhancing in un-treated
hypertensive rats as compared to normotensive rats. The significant (p < 0.001) decrease in
HDL-cholesterol was also observed. The administration of Cyperus rotundus extract at the
respective doses of 100 and 200 mg/kg or nifedipine (10 mg/kg) resulted in a significant
reduction in overall cholesterol as, compared to un-treated HTRs. The administration of
Cyperus rotundus extracts (100mg/kg and 200mg/kg) or nifedipine (10mg/kg) also resulted
in a significantly decrease in triglyceride (TG), levels as compared to un-treated hypertensive
rats (HTR). Cyperus rotundus extract (100 and 200 mg/kg) or nifedipine (10 mg/kg) also
significantly reduced the levels of LDL cholesterol as compared to HTR receiving distilled
water. Treatment with Cyperus rotundus at the dose of 200 mg/kg or nifedipine (10 mg/kg)
resulted in a significant increase in the level of HDL-cholesterol as compared to un-treated
hypertensive rats.

68
 Results & Discussion
Table 3.8 Effects of hydro-alcoholic extract of Cyperus rotundus on Lipid parameters in
Hypertensive rats.

Each value represents the mean ± SEM (n = 5). *: p <0.05 significant difference from normotensive rats.

3.9 Effects of Cyperus rotundus Hydro-alcoholic extracts on Markers of Oxidative stress

parameters in Hypertensive rats:

Treating with alcohol inducing a significanct increase (p<0.001) in the cardiac MDA
concentration, as compared to normo-tensive rats. The plant extract significantly reduced

69
 Results & Discussion
(p<0.001) MDA conc. in the heart, at doses of 100 mg/kg and 200 mg/kg as comparing to un-
treated hypertensive rats. Treatment with alcohol induced a significant decrease in catalase
activity in the heart, as compared to normotensive rats. The plant extract significantly
increased catalase activity in the heart, at the dose of 100mg/kg and 200mg/kg as compared
to hypertensive rats. Alcohol induced a significant increase of GSH conc. in the heart as
compared to normotensive rats. The extracts inducing a significant decreasing in GSH conc.
in the heart, at doses of 100mg/kg and 200mg/kg as compare to un-treated hypertensive rats.
Ethanol induced significant increase in SOD activity in the cardiac as compared to
normotensive rats.

Table 3.9.Effect of MALONDIALDEHYDE (MDA) activity in the heart of hypertensive

rats:

70
 Results & Discussion

Each value represents the mean ± SEM (n = 5). *: p <0.05 significant difference from normotensive rats.

3.10 Effect of SUPEROXIDE DISMUTASE (SOD) activity in the heart of hypertensive

rats:

PARAMETERS SOD (Unit /mg protein)

NTR 49.86 ± 4.54

HTR 21.06 ± 1.90

Nif.10 49.37 ± 4.48*

C.R 100 38.58 ± 3.51*

C.R 200 32.80 ± 2.98*

71
 Results & Discussion

Each value represents the mean ± SEM (n = 5). *: p <0.05 significant difference from normotensive rats.

3.11 Effect of CATALASE (CAT) activity in the heart of hypertensive rats:

PARAMETERS CATALASE(Unit /mg protein)

NTR 8.97± 0.80

HTR 2.17 ± 0.18

Nif.10 12.06 ± 1.08*

C.R 100 5.03 ± 0.45*

C.R 200 3.98 ± 0.21*

72
 Results & Discussion

Each value represents the mean ± SEM (n = 5). *: p <0.05 significant difference from normotensive rats.

3.12 Effect of GLUTATHIONE (GSH) activity in the heart of hypertensive rats:

CATALASE GSH (Mm/g tissue)

NTR 1.93 ± 0.16

HTR 1.25 ± 0.10

Nif 10 1.80± 0.15*

C.R 100 1.60 ± 0.14*

C.R 200 0.58 ± 0.22*

73
 Results & Discussion

Each value represents the mean ± SEM (n = 5). *: p <0.05 significant difference from normotensive rats.

74
 Results & Discussion
3.10 DISCUSSION

The antihypertensive affects of the Hydro-alcoholic extracts of, Cyperus rotundus was
evaluated in ethanol-induced hypertensive rats. Ethanol has been showing to promoting
hemodynamic disorders influences that reason of hypertension [134]. In the present
investigation, organization of ethanol 40 degree at the portion of 5 g/kg/day for 5 weeks
significantly about a huge increment in systolic, diastolic, and mean blood force and also,
cardiac rhythm. A few instruments are in charge of introducing this HTA [135]. Chronic
ingestion of liquor has been showing to actuate cardiovascular improper functioning and in
this way is the major contributory factor of high BP. [136] announced that ethanol has
complex direct vascular effects, including basal narrowing and noticeable flexible lamellae
segmentation, which influence vessels' flexibility. Vascular improper functioning because of
ethanol may initiate changes in the relaxant limit and lowering the endothelial releasing of
nitric oxide. Organization of Cyperus rotundus extracts or nifedipine fundamentally lowering
blood force in HTR. These outcomes propose that the hydro-alcoholic extracts of, Cyperus
rotundus have an anti-hypertensive influence that could be because of an activity on
peripheral resistance. In un-treated hypertensive rats, the ehhancing in blood force was joined
by an eduction of weight gain and a resulting decreasing in tissue protein levels in the
heart.This could-be because of the harmful affects brought about by liquor in the cells [137].
As per those authors, liquor preventing the absorption of supplements by kills the cells that
spread the stomach and digestive tract. Liquor likewise preventing the oxidation of
supplements by decreasing the flow of stomach related chemicals by the pancreas [138]. Like
nifedipine, the hydro-alcoholic concentrate of Cyperus rotundus essentially enhancing the
levels of protein in these various organs and furthermore animated weight gain toward the
finish of treatment when compared with hypertensive rats accepting purified water. These
outcomes propose that Cyperus rotundus ready to protecting the structural morality of cells
against the attacks of dangerous atoms got from the digestion of ethanol.

Dyslipidemia is a huge and autonomous hazard factor of cardiovascular disorder. Ethanol

treated rats demonstrated a significantly reducing in HDL-cholesterol and increment in total
cholesterol, triglycerides, and LDL-cholesterol levels just as the atherogenic record. These
findings are in agreement with results obtain by some authors who reported that
hypercholesterolemia and dyslipidemia are associated with the pathogenesis of elevated BP

75
 Results & Discussion
initiated by chronic ethanol and sucrose admission or Cyperus rotundus [139,140,141],
dyslipidemia is a primary hazard factor for atherosclerosis that advances enhancing vascular
resistance and adds to increasing circulatory pressure.

This is proper in our examination by the abnormal amounts of the atherogenic record. Like
nifedipine, the hydro-alcoholic concentrate of Cyperus rotundus essentially decreases serum
triglyceride, in total cholesterol, LDL cholesterol, and atherogenic record levels
notwithstanding an expansion in HDL-cholesterol levels. These outcomes recommend that
Cyperus rotundus concentrate contains substances that could correct dyslipidemia in liquor
instigated hypertension. Chronic disorder like hypertension is constantly joined by oxidative
pressure in responsible of their complications.

High utilization of liquor is related with oxidative pressure and enhancing the levels of free
radicals, which have been accounted for to assume a significant job in the pathogenesis of
hypertension Analysis of oxidative pressure parameters showing that malondialdehyde levels
increasing fundamentally in the heart of hypertensive rats accepting purified water and
ethanol.The abnormal state of MDA, the most significant marker of lipid peroxidation in
HTR, is an indication of cell membrane adjustment, consequently advancing the lacking of
proteins into the circulation system [141].

The significantly decreasing in reducing glutathione levels in the investigated organs in

hypertensive rats The digestion of liquor is closely identified with the generation of receptive
oxygen species prompting oxidative pressure Reduced glutathione is an endogenous, non-
enzymatic anti-oxidants in its oxidized structure; it acts by straightforwardly catching
responsive oxygen species [142].

The reduction in GSH examine in the present study is obviously related to ethanol-induced
oxidative stress, which is characterised through the production of acetaldehyde (toxic
metabolite) and other reactive molecules in the cell [143]. The treatment of HTR with the
plant extracts significantly enhancing the conc. of GSH and decreasing that of, MDA in
tissue, suggesting that, Cyperus rotundus extracts canbe act by directly trapping free-radical
and decreasing their production, therefore limiting the use of GSH. These resulting are in
conformity with some-works [144] which showed that the antioxidant activity of some plant
extracts is related to the enhancing in GSH, level. Our results also showing that ethanol

76
 Results & Discussion
significantly decrease the action of superoxide dismutase (SOD) and also that of as, catalase.
SOD and catalase are important enzymes, which protect cells against free radical injury
mediated by O2 – and H2O2 . The decrease in their activity maybe because of the, excessive
ROS production The hydro-alcoholic extract of, Cyperus rotundus as well as nifedipine
improved the anti-oxidant status in HTR. These results suggest that, Cyperus rotundus had an
antioxidant affect that protecting the tissues from the deleterious effects of free-radicals
resulting from the metabolism of ethanol.

3.11 Conclusion

Cyperus rotundus hydro-alcoholic extract exhibited an antihypertensive action in ethanol-

induced hypertension. This action maybe connected to the anti-oxidative and lipid lowering
affect. This was associated with improvement of heart functions. These properties justify the
empirical use of Cyperus rotundus in the treating of high blood pressure. More study
required tobe done in order to identify the compounds isolated in the extracts.

77