Pharmacological assessment of the aqueous extract of rose oil waste from Rosa x

damascena Herrm cultivated in Georgia

Karen Mulkijanyan 2, Natela Gogitidze 2, Marine Sulakvelidze 2, Nadezhda Mushkiashvili 2, Zhana Novikova 2
and Lasha Mskhiladze 1, *
1 Direction of Pharmacognosy and Botany, Faculty of Pharmacy, Tbilisi State Medical University, 33 Vazha Pshavela ave,
Tbilisi, 0186, Georgia.
2 Department of Preclinical Pharmacological research, TSMU I.Kutateladze Institute of Pharmacochemistry, 36

P.Sarajishvili st, 0159, Tbilisi, Georgia.

World Journal of Biology Pharmacy and Health Sciences, 2021, 07(01), 001–008

Publication history: Received on 29 May 2021; revised on 01 July 2021; accepted on 03 July 2021

Article DOI: https://doi.org/10.30574/wjbphs.2021.7.1.0069

Abstract
Among Rosaceae family's most popular and important plants Rosa x damascena Herrm. holds one of the top places due
to its centuries-long application in perfumery, cosmetics, aromatherapy and medicine. Despite this, the chemical and
pharmacological study of different products obtained from R. damascena still does not lose its relevance. Currently,
considerable attention is paid to the valorization of the waste from rose oil production in order to fully utilize valuable
physiologically active constituents. The present study provides data on some pharmacological properties of the aqueous
extract of rose oil waste from the R. damascena cultivated in Georgia. In particular, evaluation of gastro- and
hepatoprotective activity of the aforesaid extract has been carried out in animal models and revealed its efficacy. The
extract appeared to dose dependently stimulate leucopoiesis (up to 75%) and prevent ethanol-induced gastric injury
(20-80%). As well, the extract ameliorated hepatoxic effects of carbon tetrachloride by 63%.

Keywords: Rosa x damascena Herrm; Aqueous extract; Flavonoids; Gastroprotective; hepatoprotective

1. Introduction
Rosa x damascena Herrm - one of the most famous representatives of the genus Rosa L., comprising more than 400
species [1] [2] has been introduced and grown in different countries, owing to its excellent decorative features and the
presence of valuable product - essential oil in the flower petals. Since ancient times, rose oil and rose water have been
used as raw material for cosmetics, perfumery, aromatherapy, and diverse medical purposes [3].

The yield of the rose oil from flower petals is quite low (0.030–0.045%), and oil production process generates a
significant amount of waste (solid residues and waste water) that still contains biologically active compounds.
Polyphenols, flavonoids and polysaccharides were found in wastes from the rose oil industry [4-6]. On the other hand,
the same constituents determine various pharmacological activities of R. damascena including but not limited to anti-
inflammatory [7] [8], antioxidant [7] [9] [10] [11], and analgesic [12].

Recently, industrial production of rose oil from local cultivar of R. damascena have been established in Kakheti region
of Georgia, in present study, we attempted to evaluate some pharmacological properties of the aqueous extract (RDE)
of rose oil waste from the R. damascena Georgian cultivar. It is established, that the predominant constituents of RDE


Corresponding author: Lasha Mskhiladze
Direction of Pharmacognosy and Botany, Faculty of Pharmacy, Tbilisi State Medical University, 33 Vazha Pshavela ave, Tbilisi, 0186,
Georgia.
Copyright © 2021 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0.
 World Journal of Biology Pharmacy and Health Sciences, 2021, 07(01), 001–008

are represented by flavonoids [13] [14]. Flavonoids are of the most diverse and widespread groups of plant secondary
metabolites well known for having a broad spectrum of biological activity, e.g., anti-inflammatory, anticarcinogenic,
antidepressant, antibacterial, antimutagenic, anti-HIV, etc. [8] [15]. Due to generally known ability of flavonoids to
scavenge reactive oxygen species and suppress the production of pro-inflammatory factors [16] [17] we did not repeat
these assays and focused on in vivo experiments. In particular, the evaluation of gastro- and hepatoprotective activity
of the aforesaid extract, as well as its leucopoietic properties, was carried out in corresponding animal models.

2. Material and methods

2.1. Drugs and chemicals

Carbon Tetrachloride (CAS Number: 56-23-5), Absolute Ethanol (CAS Number: 64-17-5), Pentobarbital sodium salt
(CAS Number: 57-33-0) used in the experiments were obtained from Sigma Aldrich (USA). Cyclophosphamide and
Sodium Chloride 0.9% were purchased from LLC “Aversi”, Georgia. Plant raw was supplied by Pirose LTD, Georgia.

2.2. Aqueous extract of R. damascena flower petals (RDE)

R. damascena flower petals were processed by hydro distillation according to the standard procedure described in the
European Pharmacopoeia (Ph. Eur. 2008). Waste water was concentrated using rotary vacuum evaporator below 40ºC,
frozen in a layer of 10 mm in the Petri dishes at -20 °C for 12 h, and vacuum dried to constant weight at -90 °C under
3.33 Pa. Finally, the dried material was powder-grinded and stored in vacuum desiccator until further use.

2.3. Experimental

2.3.1. Animals
Inbred white mice weighing 28 ± 2 g (n = 40) were obtained from the animal house of Tbilisi State Medical University I.
Kutateladze Institute of Pharmacochemistry and quarantined for 1 week in the Department of Preclinical
Pharmacological Research of above Institute. Animals were kept under standard conditions (temperature 20 ± 2℃,
humidity 55-65%, 12/12-hour light/darkness cycle, granulated food - 4 g/animal/day, water ad libitum). All
experiments were carried out in accordance with the requirements of the EU Directive 2010/63. Research protocol was
authorized by the Tbilisi State Medical University Ethics Committee on Animal Research (approval # AP-52-2021).

2.3.2. Determination of LD50

The study was conducted using Lorke method [18] modified by Akhila et al. [19]. In brief, ten groups of three animals
each were used. A range of doses of the RDE was tested, starting from the lowest dose (10 mg/kg, intraperitoneally),
with increments of 2. The treated animals were monitored for 24 h for mortality after the administration of each dose.
The geometric mean of the highest dose which did not killed any mice and the lowest dose which led to death of all
animals these two doses was taken as the median lethal dose (LD50).

2.3.3. Hepatoprotective activity (modificalion of CCI4-induced prolongation of pentobarbital sleeping time in mice)
The hepatoprotective effect of RDE was evaluated in a modified model of CCl4-induced liver damage (potentiation of
pentobarbital sleeping time). Experiments were performed on 30 mice of both sexes weighing 26–32 g (10 animals in a
group). Group II (experimental) - RDE was administered intraperitoneally in a dose 50 mg/kg for 3 days. Within 1 hour
after the third injection and also on the next day, CCl4 (diluted 1:1 with olive oil) in a dose of 1 ml/kg was injected
subcutaneously. Then the same dose of RDE was given for another 6 days (9 in total). Group II (negative control)
received saline (0.1 ml, i.p.) and the same dose of CCl4. Group I (intact control) – was given only saline (0.1 ml, i.p.) for 9
days and sham injected on day 3-4. On day 10 pentobarbital (45 mg/kg i.p.) was injected to mice of all groups and
duration of pentobarbital sleeping time, defined as a time between loss and recovery of righting reflex, was recorded.
Hepatoprotective efficacy was calculated by the following formula:

∆𝑇con − ∆𝑇exp
E = × 100
∆𝑇con

where Tcon and Texp are the mean differen ces between sleeping time in groups II and III and II and I, correspondingly.

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2.3.4. Gastroprotective activity (Ethanol induced ulcer model)

The experiment was carried out in accordance with the method described by Adinortey et al. [20]. In brief, 24 outbred
mice were randomly distributed in three groups of animals, each consisting of eight mice. 24 hours prior to the
experiment the access to food was restricted, and animals were relocated in cages with raised floors of wide wire mesh
to prevent coprophagy. During the fasting period, all mice received a nutritive solution of 8% sucrose in 0.2% NaCl to
avoid excessive dehydration. On day 2 absolute ethanol was given orally (1 ml/100 g) to all animals. RDE in a dose of 50
mg/kg, i.p. (Group III) or 100 mg/kg, i.p. (Group II) was given 1 hour prior the ethanol administration. Mice of control
group (Group I) got 0.2 ml of saline. Animals were euthanized by CO2 inhalation 1 hour after the ethanol administration.
The stomachs were immediately removed, opened along the great curvature, rinsed consecuently with water and 10%
formalin solution which contains about 4% formaldehyde w/v, fixed on white EPS foam board, and digitally
photographed. Ulcerative lesions were measured using Image-J software and macroscopic ulcer index (MUI) was
calculated for each stomach according to the following scale: 0 - no lesions; 1 - single petechial lesions (n <10); 2 -
multiple (n≥10) petechial or short linear (≤ 2 mm) haemorrhagic lesions; 3 - long (> 2 mm) linear haemorrhagic lesions;
4 - continuous linear haemorrhagic lesions along the entire length of the glandular part of stomach. The efficacy of RDE
expressed as percentage of ulcer inhibition (% I) was estimated on the basis of the MUI and calculated using the formula:

𝑀𝑈𝐼𝑐 − 𝑀𝑈𝐼𝑇
%I = × 100
𝑀𝑈𝐼𝑇

where MUIC and MUIT are macroscopic ulcer indexes in control and test groups, respectively.

2.3.5. Leucopoietic activity (cyclophosphamide-induced leukopenia in mice)

32 mice were randomly divided in three groups (8 animals in each). Group I – intact control; group II – negative control
(only cyclophosphamide); group III (cyclophosphamide + RDE 20 mg/kg); group IV (cyclophosphamide + RDE 50
mg/kg). Acute leukopenia was induced in mice of groups II-IV by a single intraperitoneal injection of cyclophosphamide
at a dose of 350 mg/kg. Starting from day 2 after the administration of cyclophosphamide groups II and III were given
RDE orally for 5 days. Blood sampling was performed on day 1 (basal level), day 2 (to estimate the rate of leukopenia)
and after the completion of treatment (day 8 from the beginning of the experiment). Blood samples were collected under
anaesthesia with pentobarbital sodium (45 mg/kg, i.p.) from abdominal vena cava in accordance with sampling protocol
[21], and then mice were sacrificed by decapitation. Total white blood cell (WBC) counts were performed manually for
each sample using a Neubauer chamber and microscopic examination of Romanowsky-stained smears with 70X
objective [22].

2.3.6. Statistical analysis

All values were expressed as mean ± SEM. Statistical analysis of the experimental data was performed using Student’s
t-test [23]. Differences were considered significant at p < 0.05.

3. Results and discussion

3.1. Acute toxicity studies

In the first phase, mice were divided into three groups of 3 mice each and treated with the RDE in a dose of 10, 500 and
5000 mg/kg body weight intraperitoneally. Animals were observed for 24 h for signs of toxicity, mortality and general
behaviors. In the second phase, twenty-one mice were divided into 7 groups of three animals each that were
administered with the RDE at doses interval between 10 and 650 mg/kg i.p. and median lethal dose (LD50) was
calculated. LD50 of RDE appeared to be 350 mg/kg.

3.2. Ethanol induced ulcer model

In untreated animals, administration of absolute ethanol induced marked gross mucosal lesions, including full length
haemorrhagic streaks along the longitudinal axis of the glandular part of stomach and petechial lesions. (Fig.1, a I). In
mice given 100 mg/kg i.p. RDE none or only single petechial lesions were present (Fig.1, a II), whereas in animals
pretreated with 50 mg/kg RDE mainly partial length longitudinal hemorrhagic streaks were observed (Fig.1, a III).
Correspondingly, the MUI was significantly reduced in animals pretreated with RDE 100mg/kg (MUI=0.38±0.31;
%I=83) compared to RDE 50 mg/kg (MUI=2.00±1.1; %I=22) and untreated mice (MUI=2.57±1.62, Fig. 1, b, c).

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Since ethanol induced hemorrhagic damage on the gastric mucosa is commonly associated with oxidative stress [24]
[25], it is natural to assume that natural compounds with antioxidant activity can prevent or reverse such damage.
Among plant secondary metabolites, flavonoids are claimed to have various pharmacological activities in the
gastroprotective domain, including anti-secretory, cytoprotective, antihistaminic and antioxidant characteristics [26-
30]. On the other hand, it should be taken into consideration that the predominant constituents of the RDE are
flavonoids, mainly quercetin glycosides. Hence it is likelihood that the observed gastroprotective effect of RDE may be
attributed to antioxidative activity of its flavonoid content [24] [26] [31].

Figure 1 Gastroprotective effect of RDE. A – Macroscopic view of ethanol induced ulcer lesions in control (I) and RDE
treated (II and III) mice; B – Macroscopic ulcer index (MUI); C - Efficacy of RDE (%I). Each value represents mean ± SEM
of 6 animals; * - p<0.05 vs negative control

3.3. CCI4-induced prolongation of pentobarbital sleeping time

In in vivo investigations, liver injury caused by CCl4, the most well-studied system of xenobiotic-induced hepatotoxicity
[32] [33] [34], is a common model for evaluating pharmacological anti-hepatotoxic/hepatoprotective activities [35] [36]
[37].

Figure 2 RDE hepatoprotective effect in CCl4 treated mice. Each value represents mean ± SEM of 6 animals. # - p <
0.05 vs CCl4 group; * p < 0.01 vs control group

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In particular, liver damage causes an increase in pentobarbital induced sleeping time after carbon tetrachloride
poisoning. As pentobarbital is metabolized solely in the liver [38] sleep duration indicates the intensity of hepatic
metabolism. Sleeping time is entirely or partially restored in the presence of a hepatoprotective medication [39] [40]
[41] [42]. In our experiment CCI4, expectedly, more than doubled the duration of pentobarbital-induced sleeping time,
whereas the RDE treatment at a dose of 50 mg/kg reduced the hepatotoxic impact of CCI4 resulting in a 63% reduction
in sleep duration (Fig.2).

Most likely, the observed hepatoprotective effects of PDE may be related to the flavonoids present in the extract, since
these phytochemicals, are claimed to have been implicated as hepatoprotectors on CCl4 induced toxicity [43] [44] [45].

3.4. Leucopoietic activity (cyclophosphamide-induced leukopenia in mice)

The basal count of total WBC was within the normal range for mice in all groups. On day 2 after the cyclophosphamide
administration approximately 80% reduction in total WBC was observed in groups II-IV (Table 1). 5 day RDE treatment
led to the recovery of total WBC up to 64% and 75% of basal level in RDE treated groups III and IV, respectively (Fig.3).

Table 1 Ameliorative effect of RDE against cyclophosphamide (CP) induced leukopenia in mice

Total WBC (*109/l)

Group
day 1 day 2 day 8
I 5.34±0.84 5.27±1.42 5.10±1.20

II 5.98±1.12 1.08±0.85 2.20±0.65

III 6.18±1.46 1.24±0.38 3.95±1.02*

IV 5.47±1.34 1.15±0.44 4.10±0.86*

I – intact control; II - negative control (CP 350 mg/kg); III - CP+RDE 20 mg/kg ; IV - CP+RDE 50 mg/kg; * - p<0.05 vs negative control

Figure 3 Rate of leucopenia (% reduction of total WBC) in CP and RDE treated animals. Each value represents mean ±
SEM of 6 animals

Presumably, the observed effect is associated with the presence of flavonoids in RDE as complex flavonoid-containing
preparations are said to reverse leucopenia caused by cyclophosphamide when given in combination with it [47] [48]
[49] [50]. On the other hand, limitation of the study of such preparations is a lack of relation of the effect to the particular
molecule in the complex compound. Therefore, further study is needed to determine the constituents of the RDE
responsible for the leukopoietic activity.

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4. Conclusion
The data of presented study indicate some beneficial effects of the RDE. In particular, it significantly protected against
mucosal damage induced by absolute ethanol, alleviated carbon tetrachloride hepatotoxicity and contributed the
recovery of white blood cells after cyclophosphamide treatment. It is notable that all of aforesaid disorders are closely
linked with oxidative damage, and the observed effects may be attributed to predominant flavonoidal constituents of
the RDE, including, but not limited, to quercetin glycosides. These results clearly indicate that further experimentation
is needed to determine the active principles of RDE responsible for the observed pharmacological activity, and to
elucidate the exact mechanisms of their action.

Compliance with ethical standards

Acknowledgments
The authors are thankful to the Tbilisi State Medical University Iovel Kutateladze Institute of Pharmacochemistry for
the support and express their gratitude to Pirose LTD for supplying the raw plant material for this study.

Disclosure of conflict of interest

The authors declare no financial or any other conflicts of interest in this work.

Statement of ethical approval

The animal research protocol was approved by the Tbilisi State Medical University Ethics Committee on Animal
Research (registration #AP-52-2021).

References
[1] WFO (2021): Rosa × damascena Herrm. Available from http://www.worldfloraonline.org/taxon/wfo-
0001011190.
[2] Mahboubi M. Rosa damascena as holy ancient herb with novel applications. Journal of Traditional and
Complementary Medicine. 2016; 6: 10-16.
[3] Castleman M. The New Healing Herbs: The Ultimate Guide Nature's Best Medicines. NY, Bantam Book. 2001.
[4] Olech M, Nowak R, Nowacka N, Pecio Ł, Oleszek W, Los R, Malm A, Rzymowska J. Evaluation of rose roots, a post-
harvest plantation residue as a source of phytochemicals with radical scavenging, cytotoxic, and antimicrobial
activity. Ind Crops Prod. 2015; 69: 129–136.
[5] Slavov A, Vasileva I, Stefanov L, Stoyanova A. Valorization of wastes from the rose oil industry. Rev Environ Sci
Biotechnol. 2017; 16: 309–325.
[6] Sabahi Z, Farmani F, Mousavinor E, Moein M. Valorization of Waste Water of Rosa damascena Oil Distillation
Process by Ion Exchange Chromatography. The Scientific World Journal. 2020.
[7] Dossi CG, González-Mañán D, Romero N, Silva D, Videla LA, Tapia GS. Anti-oxidative and anti-inflammatory effects
of Rosa Mosqueta oil supplementation in rat liver ischemia-reperfusion. Food Funct. 2018; 9(9): 4847-4857.
[8] Shukla R, Vikas P, Gautam V, Santram L. Role of Flavonoids in Management of Inflammatory Disorders. In: Watson
R, Preedy V, eds. Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases. 2nd
ed. Academic Press. 2019; 293-322.
[9] Ng TB, He JS, Niu SM, Pi ZF, Shao W, Liu F, Zhao L. A gallic acid derivate and polysaccharides with antioxidative
activity from rose (Rosa rugosa) flowers. J Pharm Pharmacol. 2004; 56(4): 537–545.
[10] Ozkan G, Sagdic O, Baydar NG, Baydar H. Antioxidant and anti-bacterial activities of R. damascena flower extracts.
Food Sci Technol Int. 2004; 10: 277–281.
[11] Wen‐ya Liu, Lin‐yu Chen, Ying‐ying Huang, Ling Fu, Le‐yuan Song, Yun‐yu Wang, Zhe Bai, Fang‐feng Meng, Yue‐
feng Bi. Antioxidation and active constituents analysis of flower residue of Rosa damascena. Chinese Herbal
Medicines. 2020; 12(3): 336-341.

6
 World Journal of Biology Pharmacy and Health Sciences, 2021, 07(01), 001–008

[12] Hajhashemi V, Ghannadi A, Hajilooa M. Analgesic and Anti-inflammatory Effects of Rosa damascena
Hydroalcoholic Extract and its Essential Oil in Animal Models. Iran J Pharm Res. 2010; 9(2): 163–168.
[13] Kumar N, Singh B, Kaul V. Flavonoids from Rosa damascena Mill. Natural Product Communications, 2006;
1(8):623-626.Ai G, Liu Q, Hua W, Huang Z, Wang D. Hepatoprotective evaluation of the total flavonoids extracted
from flowers of Abelmoschus manihot (L.) Medic: In vitro and in vivo studies. J Ethnopharmacol. 2013; 146(3):
794-802.
[14] Akram M, Riaz Z, Altaf SH, Zahid R, Said Khan F, Riaz M, Munir N, Akhter N, Zafar S, Jabeen F, Ali Shariati M, Akhtar
N, Daniyal M. Chemical constituents, experimental and clinical pharmacology of Rosa damascena: a literature
review, Journal of Pharmacy and Pharmacology. 2020; 72(2): 161–174.
[15] Romano B, Pagano E, Montanaro V, Fortunato AL, Milic N, Borrelli F. Novel insights into the pharmacology of
flavonoids. Phytother Res. 2013; 27(11): 1588-96.
[16] Chen H, Pu J, Liu D, Yu W, Shao Y, Yang G, Xiang Z, He N. Anti-Inflammatory and Antinociceptive Properties of
Flavonoids from the Fruits of Black Mulberry (Morus nigra L.). PLoS ONE. 2016; 11(4): e0153080.
[17] Chen H, Yu W, Chen G, Meng S, Xiang Z, He N. Antinociceptive and Antibacterial Properties of Anthocyanins and
Flavonols from Fruits of Black and Non-Black Mulberries. Molecules. 2017; 23(1): 4.
[18] Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol. 1983; 54(4): 275–287.
[19] Akhila JS, Shyamjith D, Alwar MC. Acute toxicity studies and determination of median lethal dose. Curr. Sci. 2007;
93: 917–20.
[20] Adinortey MB, Ansah C, Galyuon I, Nyarko A. In Vivo Models Used for Evaluation of Potential Antigastroduodenal
Ulcer Agents. Ulcers. 2013; 12.
[21] Hoff J. Methods of Blood Collection in the Mouse. Lab Animal. 2000; 29(10): 47-53.
[22] Marshall PN. Romanowsky-type stains in haemotology. Histochem J. 1978; 10: 1–29.
[23] McDonald JH. Handbook of Biological Statistics, 3rd ed. Baltimore, Sparky House Publishing. 2014.
[24] Coşkun Ö, Kanter M, Armutçu F, Çetin K, Kaybolmaz B, Yazgan Ö. Protective effects of quercetin, a flavonoid
antioxidant, in absolute ethanol-induced acut gastric ulcer. Eur J Gen Med. 2004; 1(3): 37-42.
[25] Mei X, Xu D, Xu S, Zheng Y, Xu S. Novel role of Zn(II)-curcumin in enhancing cell proliferation and adjusting
proinflammatory cytokine mediated oxidative damage of ethanol-induced acute gastric ulcers. Chem Biol
Interact. 2012; 197: 31–39.
[26] Kahraman A, Erkasap N, Köken T, Serteser M, Aktepe F, Erkasap S. The antioxidative and antihistaminic
properties of quercetin in ethanol-induced gastric lesions. Toxicology. 2003; 183(1-3): 133-42.
[27] Mota K, Dias G, Pinto M, Luiz-Ferreira A, Souza-Brito A, Hiruma-Lima C, Barbosa-Filho J, Batista L. Flavonoids
with Gastroprotective Activity. Molecules. 2009; 14: 979-1012.
[28] Sumbul S, Ahmad MA, Mohd A, Mohd A. Role of phenolic compounds in peptic ulcer: An overview. J Pharm
Bioallied Sci. Jul 2011; 3(3): 361-7.
[29] Serafim C, Araruna ME, Júnior EA, Diniz M, Hiruma-Lima C, Batista L. A Review of the Role of Flavonoids in Peptic
Ulcer (2010-2020). Molecules. 2020 Nov 20;25(22):5431. https://doi.org/10.3390/molecules25225431. PMID:
33233494; PMCID: PMC7699562Alqasoumi S. Carbon tetrachloride-induced hepatotoxicity: Protective effect of
'Rocket' Eruca sativa L. in rats. Am J Chin Med. 2010; 38(1): 75-88.
[30] Zhang W, Lian Y, Li Q, Sun L, Chen R, Lai X, Lai Z, Yuan E, Sun S. Preventative and Therapeutic Potential of
Flavonoids in Peptic Ulcers. Molecules. 2020; 25(20): 4626.
[31] Balan T, Sani MH, Mumtaz Ahmad SH, Suppaiah V, Mohtarrudin N, Zakaria ZA. Antioxidant and anti-inflammatory
activities contribute to the prophylactic effect of semi-purified fractions obtained from the crude methanol
extract of Muntingia calabura leaves against gastric ulceration in rats. J Ethnopharmacol. 2015; 22(164):1-15.
[32] Slater T, Cheeseman K, Ingold K, Rice-Evans C, Sies H. (Carbon Tetrachloride Toxicity as a Model for Studying
Free-Radical Mediated Liver Injury [and Discussion]. Philosophical Transactions of the Royal Society of London.
Series B, Biological Sciences. 1985; 311(1152): 633-645.
[33] Weber LW, Boll M, Stampfl A. Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a
toxicological model. Crit Rev Toxicol. 2003; 33(2): 105-36.

7
 World Journal of Biology Pharmacy and Health Sciences, 2021, 07(01), 001–008

[34] Toriumi K, Horikoshi Y, Yoshiyuki Osamura R, et al. Carbon tetrachloride-induced hepatic injury through
formation of oxidized diacylglycerol and activation of the PKC/NF-κB pathway. Lab Invest. 2013; 93: 218–229.
[35] Gilani AUH, Janbaz KH. Preventive and curative effects of Berberis aristata Fruit extract on paracetamol- and
CCl4-induced hepatotoxicity. Phytother Res. 1995; 9: 489-494.
[36] Al-Harbi NO, Imam F, Nadeem A, Al-Harbi MM, Iqbal M, Ahmad SF. Carbon tetrachloride-induced hepatotoxicity
in rat is reversed by treatment with riboflavin. International Immunopharmacology. 2014; 21(2): 383-388.
[37] Almatroodi SA, Ahmad Almatroudi A, Anwar S, Babiker AY, Khan AA, Alsahli MA, Rahmani AH Antioxidant, anti-
inflammatory and hepatoprotective effects of olive fruit pulp extract: in vivo and in vitro study, Journal of Taibah
University for Science. 2020; 14(1): 1660-1670.
[38] Ossenberg FW, Peignoux M, Bourdiau D, Benhamou JP. Pentobarbital pharmacokinetics in the normal and in the
hepatectomized rat. J Pharmacol Exp Ther. 1975; 194(1): 111-116.
[39] Montilla M, Cabo J, Navarro M, Risco S, Jiménez J, Aneiros J. The protective and curative action of Withania
frutescens leaf extract against CCl4-induced hepatotoxicity, Pytotherapy research. 1990; 4(6): 212–215.
[40] Alqasoumi S. Carbon tetrachloride-induced hepatotoxicity: Protective effect of 'Rocket' Eruca sativa L. in rats. Am
J Chin Med. 2010; 38(1): 75-88.
[41] Dighe SB. Effect of Oxalis corniculata extracts on Pentobarbital-induced sleeping time in carbon
tetrachlorideinduced hepatotoxic rats. J Appl Pharm. 2018; 10: 29.
[42] Daniel EP, Vijayakumar AR, Atlee WC, Ilavarasan R, Sivakumar M. Effect of carbon tetrachloride-induced hepatic
injury on Luffa acutangula (var.) amara. Phcog Res. 2020; 12(3): 278-284.
[43] Aniya Y, Shimabukuro M, Shimoji M, Kohatsu M, Gyamfi MA, Miyagi C, Kunii D, Takayama F, Egashira T.
Antioxidant and hepatoprotective actions of the medicinal herb Artemisia campestris from the Okinawa Islands.
Biol Pharm Bull. 2000; 23(3): 309-12.
[44] Kim SM, Kang K, Jho EH et al. Hepatoprotective effect of flavonoid glycosides from Lespedeza cuneata against
oxidative stress induced by tert-butyl hyperoxide. Phytother Res. 2011; 25(7): 1011–1017.
[45] Tong J, Yao X, Zeng H, Zhou G, Chen Y, Ma B, Wang Y. Hepatoprotective activity of flavonoids from Cichorium
glandulosum seeds in vitro and in vivo carbon tetrachloride-induced hepatotoxicity. J Ethnopharmacol. 2015;
174: 355-63.
[46] Nguyen TP, Tran CL, Vuong CH, Do THT, Le TD, Mai DT, Phan NM. Flavonoids with hepatoprotective activity from
the leaves of Cleome viscosa L. Nat Prod Res. 2017; 31(22): 2587-2592.
[47] Weber K, Mengs U, Schwarz T, Becker H, Lentzen H. Stimulation of neutropoiesis by a special standardized
mistletoe preparation after cyclophosphamide chemotherapy in mice. Arzneimittelforschung. 1996; 46(12):
1174-1178.
[48] Mahmoud AM. Hesperidin protects against cyclophosphamide-induced hepatotoxicity by upregulation of PPARγ
and abrogation of oxidative stress and inflammation. Canadian Journal of Physiology and Pharmacology. 2014;
92(9): 717–724.
[49] Panchenko AV, Fedoros EI, Pigarev SE, Maydin MA, Gubareva EA, Kireeva GS, Tyndyk ML, Kuznetsova AI,
Nekhaeva TL, Danilova AB, Baldueva IA, Anisimov VN. The Effect of Polyphenolic Composition BP-C3 on the
Efficacy and Hematological Toxicity of Cyclophosphamide in the Chemotherapy of Mice Bearing Soft Tissue
Sarcomas Induced by Benzo[a]pyrene. Integr Cancer Ther. 2019; 18: 1-13.
[50] Wang L, Li H, Shen X, Zeng J, Yue L, Lin J, Wu, J. Elucidation of the molecular mechanism of Sanguisorba Officinalis
L. against leukopenia based on network pharmacology. Biomedicine & Pharmacotherapy. 2020; 132: 110934.