全 文 ： 240 Chin J Nat Med May 2013 Vol. 11 No. 3 2013 年 5 月 第 11 卷 第 3 期

Chinese Journal of Natural Medicines 2013, 11(3): 0240−0244
doi: 10.3724/SP.J.1009.2013.00240
Chinese
Journal of
Natural
Medicines






Anti-anxiety activity of Stachys tibetica Vatke
Dinesh Kumar, Zulfiqar Ali Bhat *, Vijender Kumar, W Y Raja, M. Y. Shah
Department of Pharmaceutical Sciences, University of Kashmir, Srinagar 190006, India
Available online 20 May 2013
[ABSTRACT] AIM: S. tibetica Vatke is a herb distributed in the tropical and subtropical regions of the world, including Tibet, China,
and India. In India it is found in the cold desert regions of Kargil, Ladakh Valley, and in the mountains of Himachal Pradesh. The tradi-
tional practitioners in the Kargil and Ladakh use the natural medicine Stachys tibetica for the treatment of various mental disorders and
phobias. The present study is aimed at evaluating the anxiolytic effects of the methanolic extract of the root, stem, leaf, and whole plant
material of Stachys tibetica Vatke in rats. METHODS: Powdered materials (1 kg) of each plant part were subjected to extraction in a
Soxhlet apparatus with methanol (95%); to yield 12.8%, 8.3%, 17.2%, and 19.6% W/W extractives, respectively. Extracts were evalu-
ated for their anxiolytic effects using the elevated plus maze (EPM) test in rats. RESULTS: In the present study, it was found that the
methanolic extracts (200 and 400 mg·kg−1) of the root, stem, leaf and whole plant of Stachys tibetica Vatke and diazepam (DZ) increased
the time spent and the number of entries in the open arm significantly (**P < 0.01), while they decreased the time spent and the number
of entries in the closed arm. At the same time, all of the extracts and DZ decreased the time spent at the center of the maze (latency),
along with closed arm returns. The head dip counts increased significantly in the rats treated with DZ, SMR400, SML400 and
SMW400 in the open arm of EPM, which was a sign of reduction anxiety. The DZ and SMW did not show the fecal bolus, while other
groups had reduced fecal bolus (**P < 0.01) as compared to control. These allied parameters helped to assess the anxiolytic potential of
Stachys tibetica Vatke. Whole plant and leaf materials have shown the maximum activity, the root intermediate while the stem had the
least anxiolytic activity (*P < 0.05, **P < 0.01) in EPM. CONCLUSION: The results strongly justify the use of this plant for the treat-
ment of anxiety. Further studies are in progress in this laboratory to isolate and identify the components responsible for the anxiolytic
activity and the mechanism of action involved.
[KEY WORDS] Stachys tibetica Vatke; Lamiaceae; Anxiolytic; Elevated plus maze test
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2013)03-0240-05

1 Introduction
Anxiety is a cardinal symptom of many psychiatric dis-
orders and an almost inevitable component of many medical
and surgical conditions. Indeed, it is a universal human emo-
tion, closely allied with appropriate fear, and presumably
serving pyschobiologically adaptive purposes [1]. Although
many drugs are available in allopathic medicine to treat anxi-
ety disorders, like benzodiazepine, serotonin inhibitors, etc.
they produce various systemic side effects or exhibit toler-
ance upon chronic use. In natural or traditional medicine,
many plant products have been claimed to be free from side
effects and to be less toxic than synthetic drugs. India and

[Received on] 18-May-2012
[Research funding] This project was supported financially by Uni-
versity of Kashmir, Jammu and Kashmir and UGC.
[*Corresponding author] Dr. Z. A. Bhat: Sr. Asst. Prof., Tel:
91-9419077701, 09882120308, E-mail: sharmadinesh82@gmail.com;
zabhat2000@gmail.com
These authors have no conflict of interest to declare.
China are the countries of vast biodiversity and traditional
knowledge for using natural/herbal medicines to cure many
ailments in various cultures and tribes. There has been a con-
siderable popular interest in the use of herbal products, to
treat anxiety disorder/reactions. Hypericum perforatum
commonly known as St. Johns Wort; is the most well-known
herbal product available over the counter. Recently, several
herbal constituents have been reported to possess anxiolytic
effects through animal models of anxiety [2]. The historical
use of such medicine provides the source to study the specific
plant species with the potential to be used in a particular dis-
ease.
Plants belonging to the family Lamiaceae are rich in es-
sential oils. Previous studies have shown that a number of
plants from this family, including Salvia officinalis, Salvia
elegans, Salvia reuterana, and Scutellaria baicalensis have
shown anti-anxiety activity [3]. Stachys L. (Lamiaceae) is a
large genus of herbs and shrubs comprising 300 species dis-
tributed in the temperate and tropical regions of the world.
Plants of this genus have been used in folk medicine for cen-
turies to treat genital tumors, sclerosis of the spleen, inflam-
Dinesh Kumar, et al. /Chinese Journal of Natural Medicines 2013, 11(3): 240−244
2013 年 5 月 第 11 卷 第 3 期 Chin J Nat Med May 2013 Vol. 11 No. 3 241

matory diseases, anxiety, cough and ulcers. Stachys tibetica is
distributed in the tropical and subtropical regions of the
world, including in Tibet, China, India, etc. In India, it is
found in the cold desert regions of the Ladakh Valley and in
the mountains of Himachal Pradesh. The traditional practi-
tioners in the Ladakh, use the drug for the treatment of vari-
ous mental disorders and phobias [3-4]. In this study, a possible
anxiolytic property of S. tibetica was investigated using rats
in the elevated plus maze test. The EPM is one of the most
extensively used models for the investigation of drug effects
on anxiety-related behavior in laboratory rodents. It is based
on the aversion of rodents for open spaces, and anxiolytics
have been found to increase the proportion of time spent on
the open arms [5].
2 Materials and Methods
2.1 Plant material
The plant specimen of Stachys tibetica Vatke was col-
lected from Kargil and Ladakh, Jammu and Kashmir, India.
The plant was identified and authenticated by Dr. Zulfiqar Ali
Bhat of the Department of Pharmaceutical Sciences, Univer-
sity of Kashmir,Srinagar-190006, India and a Specimen
Voucher number-KUST01 was assigned to it.
2.2 Drying and size reduction of plant
The root, stem, leaf, and whole plant material of Stachys
tibetica were subjected to shade drying for about 1 week. The
dried plant material was further crushed to a powder and the
powder was passed through sieve No. 40 and stored in an
air-tight container for further analysis.
2.3 Extraction of plant material
The coarsely powdered materials (1 kg) of the root, stem,
leaf, and whole plant were subjected to extraction in a Soxhlet
apparatus with methanol (95%) to yield 12.8%, 8.3%, 17.2%,
and 19.6% W/W, respectively. All of the extracts (SMR, SMS,
SML and SMW) were concentrated and dried on a water bath
to obtain the respective extracts.
2.4 Animals
Albino rats (Wistar strain) of either sex weighing 150−200
g, respectively were used for studies. The albino rats were
obtained from the animal house of the Indian Institute of
Integrative Medicine−Jammu, Jammu and Kashmir, India.
They were housed in polypropylene cages with standard pel-
let chow and water ad libitum. In all other experimental sets,
ten rats were used for each dosage. The control group re-
ceived only vehicle and the positive control received standard
drug diazepam (DZ, 1 mg·kg−1, p.o.) while other groups re-
ceived the extracts. This institution is approved by the
CPCSEA, Government of India. (Approval No.801/03/ca/
CPCSEA) for carrying out animal studies and the protocol
for the present study was approved by the Institutional Ani-
mal Ethical Committee [Approval No. F-IAEC (Pharm. Sc.)
APPROVAL/2011/01], and the experiments were conducted
as per the approved protocol.
2.5 Acute toxicity study: (OECD guidelines-425, 2001)
Acute toxicity study was conducted as per the interna-
tionally accepted protocol drawn under the OECD guidelines
425 (OECD, 2001). Overnight fasted, healthy rats (n = 6)
were administered orally the methanolic extracts of the root,
stem, leaf, and whole plant material in the doses of 100, 200,
400, 800, 1 600 mg·kg−1 body weight and observed continu-
ously for 4 h and after 24 h for any abnormality and mortality.
All extracts at a dose level of 1 600 mg·kg−1 were found to be
safe. Doses of 200 and 400 mg·kg−1 were selected as the ex-
perimental dose of the extracts for anti-anxiety studies.
2.6 Drugs
Diazepam was obtained from Ranbaxy Lab. Ltd.,
HPSIDC-Baddi, Solan (India). Sodium carboxy methyl cel-
lulose was purchased from CDH-Laboratory Reagent Pvt.
Ltd. Post Box. No. 7138, New Delhi-110002 (India). Diaze-
pam and the methanolic extracts of the root, stem, leaf, and
whole plant of Stachys tibetica were suspended in a 1% so-
dium carboxy methyl cellulose solution. All the drugs were
prepared immediately before use and administered orally.
Control rats received 1% aqueous sodium carboxy methyl
cellulose solution only. The effects of the drugs were esti-
mated 60 min after drug administration. Tests were per-
formed only after the rats had been acclimatized to the ex-
perimental environment for at least 7 days. All experiments
were carried out between 09 : 00 and 16 : 00. In each ex-
periment, the apparatus was cleaned using 5% ethanol before
introducing the next animal to preclude the possible cueing
effects of odors left by previous subjects.
2.7 Anxiolytic activity
The test procedure and scoring methodology for elevated
plus-maze test have been described in detail elsewhere [6-7]. In
brief, the apparatus comprised two open arms (50 cm × 10
cm) and two enclosed arms of the same size with 40 cm high
wall arranged so that the arms of the same type were opposite
to each other with a central square of 10 cm to form a plus
sign. The apparatus was wooden and was elevated to a height
of 50 cm above floor level by a single central support. A
slightly raised edge on the open arms (0.25 cm) provided
additional grip for the animals, whereas open arm activity
was further encouraged by testing in a dimly lit room. The
experiment was conducted between 09 : 00 – 16 : 00. To
facilitate adaptation to new surroundings, rats were trans-
ported to the laboratory at least 1 h prior to testing. The trial
was started by placing an animal on the central platform of
the maze facing an open arm. Standard 5-min test duration
was used, and between subjects, the maze was thoroughly
cleaned. Rats were randomly allocated to the following
groups: vehicle control, positive control: Diazepam (DZ; 1
mg·kg−1, p.o.), SMR, SMS, SML and SMW (100 and 200
mg·kg−1, p.o.). The experiments were performed with an ob-
server unaware of the treatment of the rats inside the room.
The following parameters are classically measured in this test:
frequency and duration (s) of arm visits, separately for open
Dinesh Kumar, et al. /Chinese Journal of Natural Medicines 2013, 11(3): 240−244
242 Chin J Nat Med May 2013 Vol. 11 No. 3 2013 年 5 月 第 11 卷 第 3 期

and closed arms. A mouse was considered to have entered an
arm when all four paws were on the arm. The percentage of
entries into the open arms, the closed arms (open or closed
arm entries/total arm entries × 100; % open or closed arm
entries) and the percentage of time spent in the open or
closed arms (open or closed arm time/total arm time × 100;
% open or closed arm time) are used as traditional indices of
the anxiety level. In addition, the head dip count, raring, fecal
bolus (stool bal) and latency time was also recorded, i.e, the
time spent at the center of the maze [3].
2.8 Statistical analysis
All observations were presented as x ± SEM and were
analyzed using one-way analysis of variance (Anova) fol-
lowed by Dunnet’s (*P < 0.05, **P < 0.01). P values lower
than 0.05 were considered statistically significant.
3 Results
3.1 Effect of diazepam and the methanolic extract of different
parts of Stachys tibetica on elevated plus-maze test in rats
All of the extracts of Stachys tibetica at a dose of 200
and 400 mg·kg−1 significantly increased the percentage of
time spent and the arm entries in the open arms, and de-
creased the number of entries and duration of time spent in
the closed arms. In a similar fashion, the standard drug, di-
azepam, increased the percentage of time spent and the per-
centage of arm entries in the open arms (*P < 0.05, **P <
0.01). At the same time, all of the extracts and DZ decreased
the time spent at the center of the maze or the latency along
with closed arm returns. The head dip counts increased sig-
nificantly in the rats treated with DZ, SMR400, SML400, and



Fig. 1 Percentage of open arm time and closed arm time
Results are expressed as x ± SEM (n = 6). Control = vehicle, DZ = Diazepam (1 mg·kg−1, p.o.) as a standard drug, SMR 200 = methanol
extract of the root of Stachys tibetica (200 mg·kg−1, p.o.), SMR 400 = methanol extract of the root of Stachys tibetica (400 mg·kg−1, p.o.),
SMS 200 = methanol extract of the stem of Stachys tibetica (200 mg·kg−1, p.o.), SMS 400 = methanol extract of the stem of Stachys tibetica
(400 mg·kg−1, p.o.), SML 200 = methanol extract of the leaf of Stachys tibetica (200 mg·kg−1, p.o.), SML 400 = methanol extract of the leaf
of Stachys tibetica (400 mg·kg−1, p.o.), SMW 200 = methanol extract of the whole plant of Stachys tibetica (200 mg·kg−1, p.o.), MEW 400 =
methanol extract of the whole plant of Stachys tibetica (400 mg·kg−1, p.o.). *P < 0.05, **P < 0.01 vs control



Fig. 2 Percentage of open arm entries and closed arm entries
Results are expressed as x ± SEM (n = 6). Control = vehicle, DZ = Diazepam (1 mg·kg−1, p.o.) as a standard drug, SMR 200 = methanol
extract of the root of Stachys tibetica (200 mg·kg−1, p.o.), SMR 400 = methanol extract of the root of Stachys tibetica (400 mg·kg−1, p.o.),
SMS 200 = methanol extract of the stem of Stachys tibetica (200 mg·kg−1, p.o.), SMS 400 = methanol extract of the stem of Stachys tibetica
(400 mg·kg−1, p.o.), SML 200 = methanol extract of leaf of Stachys tibetica (200 mg·kg−1, p.o.), SML 400 = methanol extract of leaf of
Stachys tibetica (400 mg·kg−1, p.o.), SMW 200 = methanol extract of the whole plant of Stachys tibetica (200 mg·kg−1, p.o.), MEW 400 =
methanol extract of the whole plant of Stachys tibetica (400 mg·kg−1, p.o.). *P < 0.05, **P < 0.01 vs control
Dinesh Kumar, et al. /Chinese Journal of Natural Medicines 2013, 11(3): 240−244
2013 年 5 月 第 11 卷 第 3 期 Chin J Nat Med May 2013 Vol. 11 No. 3 243

Table 1 Effect of the methanol extracts of different parts of Stachys tibetica on allied parameters of the EPM test
Groups
(n = 6) Control DZ SMR200 SMR400 SMS200 SMS400 SML200 SML400 SMW200 SMW400
L 101.1 ± 9.01**
48.3 ±
4.33**
62.5 ±
6.66** 46 ± 4.11
** 52.7 ±
5.11**
46.5 ±
3.99**
57.4 ±
4.66**
46.3 ±
4.06**
58.4 ±
5.11**
40.3 ±
4.03**
HD 1 ± 0.1 4 ± 0.26** 1 ± 0.09 2 ± 0.17** 1 ± 0.11 1 ± 0.12 1 ± 0.1 3 ± 0.21** 1 ± 0.06 3 ± 0.09**
FB 6 ± 0.56** − 2 ± 0.12** 1 ± 0.09** 3 ± 0.23** 2 ± 0.19** 1 ± 0.11** 1 ± 0.1** 1 ± 0.99** −
CAR 7.4 ± 0.66** 0.7 ± 0.04** 1.1 ± 0.1** 1.2 ± 0.11** 3.4 ± 0.36** 2.5 ± 0.21** 2.1 ± 0.2** 0.9 ± 0.09** 1.5 ± 0.15** 0.8 ± 0.89**
L = Latency (Time spent at the center of maze), HD = Head dip count (Head dip from the open arm), FB = Fecal bolus (stool balls), CAR
= Closed arm returns, Results are expressed as x ± SEM (n = 6). Control = vehicle, DZ = Diazepam (1 mg·kg−1, p.o.) as a standard drug,
SMR 200 = methanol extract of the root of Stachys tibetica (200 mg·kg−1, p.o.), SMR 400 = methanol extract of the root of Stachys tibetica
(400 mg·kg−1, p.o.), SMS 200 = methanol extract of the stem of Stachys tibetica (200 mg·kg−1, p.o.), SMS 400 = methanol extract of the stem
of Stachys tibetica (400 mg·kg−1, p.o.), SML 200 = methanol extract of the leaf of Stachys tibetica (200 mg·kg−1, p.o.), SML 400 = methanol
extract of the leaf of Stachys tibetica (400 mg·kg−1, p.o.), SMW 200 = methanol extract of the whole plant of Stachys tibetica (200 mg·kg−1,
p.o.), MEW 400 = methanol extract of the whole plant of Stachys tibetica (400 mg·kg−1, p.o.). *P < 0.05, **P < 0.01 vs control

SMW400 in the open arm of EPM which was a sign of re-
duced anxiety (Table 1). The DZ and SMW did not show the
fecal bolus while other groups had reduced fecal bolus (**P <
0.01), as compared to control (Table 1). Whole plant and leaf
extracts have shown the most activity, the root an intermedi-
ate level, while the stem extract had the least anxiolytic
activity (*P < 0.05, **P < 0.01) in EPM (Figs. 1−2, Table 1).
4 Discussion and Conclusion
Animal models help to understand the information about
molecular mechanisms involved in anxiety, and are useful for
screening and for developing new medications for their
treatment that would be impossible in humans. The human
studies have established the genetic basis of anxiety, and
animal studies have been used to attempt to further clarify its
genetic determinants. The first involves the animal’s condi-
tioned responses to stressful and often painful events (e.g.
exposure to electric foot shock), and the second includes
ethologically based paradigms, and involves the animal’s
spontaneous or natural reactions (e.g. flight, avoidance and
freezing) to stress stimuli that do not explicitly involve pain
or discomfort (e.g. exposure to a novel highly illuminated test
chamber or to a predator) [8]. Numerous psychological and
brain disorder research studies have been conducted using
traditional medicinal plants in the form of specific oils, herbal
extracts, and combinations to treat specific diseases, includ-
ing anxiety, depression, insomnia, Alzheimer’s, convulsions,
Parkinson’s, etc. in an effort to discover new therapeutic
agents that lack the toxic side effects associated with the
current agents. The Elevated Plus-maze is a well-established
animal model and is currently the first choice test for anxiolytic
drugs, and has been validated for both rats and mice [9-10]. It is
based on the natural conflict between the drive to explore a
new environment and the tendency to avoid a potentially dan-
gerous area. More recently, it has been argued that the incor-
poration of a range of ethological parameters may enhance the
utility of this paradigm [5].
In the present study, it was found that the methanolic ex-
tracts all parts of Stachys tibetica and DZ increased the time
spent and the number of entries in the open arm significantly
(**P < 0.01), while they decreased the same in the closed arm.
At the same time, all of the extracts and DZ decreased the
time spent at the center of maze (latency) along with closed
arm returns. The head dip counts increased significantly in
the rats treated with DZ, SMR400, SML400, and SMW400 in
the open arm of EPM, which was a sign of reduced anxiety.
The DZ and SMW did not show the fecal bolus, while the
other groups had reduced fecal bolus (**P < 0.01) as com-
pared to the control. These allied parameters helped to assess
the anxiolytic potential of Stachys tibetica. Herefore, the be-
havioral alterations induced by the extracts in the EPM are
consistent with an anxiolytic effect, similar to that of diazepam.
In previous studies from these laboratories, the phyto-
chemical screening of successive and individual extracts of
the whole part material of Stachys tibetica was reported, and
it was found that the plant contain coumarins, flavonoids,
saponins, glycosides, steroids, terpenoids, alkaloids, pheno-
lics, and tannins, etc. The plant has good concentration of
flavonoids, saponins, and also has volatile oils (0.7%), which
may be responsible for the activity [4].
Stachys tibetica essential oil which has previously been
reported as an anxiolytic, is a mixture of terpenes and has
almost 62 constituents with aciphylline as the major con-
stituent [3], while the methanol extract contains both polar and
non-polar compounds, such as coumarins, flavonoids, sapon-
ins, glycosides, steroids, terpenoids, alkaloids, phenolics,
tannins, fats/lipids, acids, carbohydrates, proteins, etc. in all
of the extracted parts.
The extracts of different parts of Stachys tibetica had
different anxiolytic effects, and the effects may be due to the
presence of different classes of chemical constituents and
variation in their concentration. The other reason for the ef-
fects may be due to the synergism and antagonism actions
and competence towards the receptors like benzodiazepines,
serotonins, GABA, dopamines, adrenergic, cholinergic, his-
tamines, etc.
Further pharmacological investigations are required to
Dinesh Kumar, et al. /Chinese Journal of Natural Medicines 2013, 11(3): 240−244
244 Chin J Nat Med May 2013 Vol. 11 No. 3 2013 年 5 月 第 11 卷 第 3 期

identify the active constituents of Stachys tibetica responsible
for the anxiolytic effects. The results obtained from this study
justify the use of this important medicinal plant in the Indian
and Tibetan folklore medicine for the management of nervous
and cerebral disorders, including anxiety and phobias. Fur-
ther studies are in progress in this laboratory to isolate and
identify the components responsible for the anxiolytic activ-
ity and the mechanism of action involved. Results will lay a
way for the isolation of bioactive principles and new drug
discoveries for the treatment of anxiety.
Acknowledgments
Dinesh Kumar, Dr. ZA Bhat, and Prof. MY Shah would
like to thank University of Kashmir, Jammu and Kashmir and
UGC, New Delhi, India for financial assistance for this work.
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