Download Assiut university researches Phytochemical and Biological Studies

Assiut university researches
Phytochemical and Biological Studies Of
Certain Plants Belonging To Family
Acanthaceae
‫درا سة ف ي توك يم يائ ية وب يول وج ية ل ب عض ن بات ات‬
‫ال عائ لة األك ان ثاث ية‬
Marwa Abdel-Aziz Ali Fayed
‫مروه ع بدال عزي ز ع لى ف اي د‬
Mohamed Ahmed El-Shanawany, Hanaa Mohamed Sayed, Sabrin
Ragab Mohamed
‫ صاب ري ن رجب محمد‬،‫ ه ناء محمد س يد‬،‫محمد أحمد ال ش ناوى‬
Abstract:
Phytochemical and Biological Studies of Some Plants
Belonging to Family Acanthaceae Family Acanthaceae is
considered as one of the richest families with medicinal
plants. It includes about 346 genera and around 4300
species. The plants under investigation are Anisotes trisulcus
(Forssk.) Nees. & Blepharis ciliaris (L.) B.L.Burtt. both
belonging to family Acanthacaea. Anisotes trisulcus (Forssk.)
Nees.: A stiffy erect shrub with a height of 1-3.5 m. It is
endogenous to Yemen and widely distributed in the Southern
mountainous regions of the kingdom of Saudi Arabia
particularly in the Wadi Jabal Abu Hassan between Abha and
Najran. The main stem of the plant is erect, cylindrical,
monopodially branched carrying numerous branches showing
more or less long internodes. The young stem is purplishgreen in colour, while the old one has rough surface covered
with brownish cork. The branches bear opposite and
decussate leaves at the nodes. Flowers are shortly
pedunculate, 2-flowered axillary clusters. Fruit capsule, 4
seeded. Seeds are rough. Blepharis ciliaris (L.) B.L.Burtt.: A
perennial, greyish-pubescent to glabrescent ascending,
prickly herb branched from the base, reaching up to 30 cm in
height, distributed in East Tropical Africa, Saudi Arabia and
Egypt. The leaves appearing in whorls of 4-outer pairs in
each whorl often smaller, they are oblong to lanceolate,
coriacious lamina with silvery appearance and long petiole,
margins are entire to remotely spinulose, puberulous above,
with spine tipped apex. Inflorescence is a dense, strobilate
spike with purplish-blue flowers, bracteoles are linearsubulate villose. Fruit is a capsule, ovate to compressed
shining brown and glabrous with two flat seeds. Anisotes
trisulcus (Forssk.) Nees. is used as traditional herbal
medicine in the Arabian Peninsula as a treatment for all
hepatic conditions including hepatitis, jaundice, gallstone and
other hepatic disorders. It is also used as anti-diabetic,
bronchodilator, hypo-tensive and it has a local anesthetic
effect. The plant extract is used in several pharmaceutical
forms to limit tobacco consumption and to suppress appetite.
Blepharis ciliaris (L.) B.L.Burtt. seeds (roasted or crushed)
were applied on sores, wounds and boils as an antibacterial.
The seeds are also considered to be attenuant, resolvent,
diuretic, aphrodisiac and expectorant in addition to charcoal
from the roots is applied to the eyes to improve vision, hence
the Arabic name ”Kohl-el-agouz”. Previous Phytochemical
and biological studies of Acanthaceous plants especially
Anisotes trisulcus (Forssk.) Nees. and Blepharis ciliaris
(L.)B.L. Burtt. showed the isolation and identification of
diverse secondary metabolites as well as different biological
activities. Thus, it was deemed important to carry out
comprehensive phytochemical and biological investigations of
the selected plants. The present work includes the following
three parts: PART I: Phytochemical Study of Anisotes
trisulcus (Forssk.) Nees. Aerial Parts. Chapter I: Preliminary
phytochemical screening of the air dried powdered aerial
parts of Anisotes trisulcus (Forssk.) Nees. Chapter II:
Extraction, fractionation and isolation of the constituents from
Anisotes trisulcus (Forssk.) Nees. Chapter III: Identification of
the isolated compounds from Anisotes trisulcus (Forssk.)
Nees. aerial parts. PART II: Phytochemical Study of Blepharis
ciliaris (L.) B.L.Burtt. Aerial Parts. Chapter I: Preliminary
phytochemical screening of the powdered Blepharis ciliaris
(L.) B.L.Burtt.aerial parts. Chapter II: Extraction, fractionation
and isolation of the constituents from Blepharis ciliaris (L.)
B.L.Burtt.aerial parts. Chapter III: Identification of the isolated
compounds from Blepharis ciliaris (L.) B.L.Burtt. aerial parts.
PART III: Biological Studies of Anisotes trisuclus (Forssk.)
Nees. and Blepharis ciliaris (L.)B.L.Burtt. Aerial Parts. 1Antioxidant Activity. 2- Acute Toxicity Study. 3- Brine-Shrimp
Assay (Cytotoxic Activity). 4- Anti-inflammatory Activity. 5Anti-malarial Activity. 6- Anti-hyperglycaemic Activity. PART I
Phytochemical Study of Anisotes trisulcus (Forssk.) Nees.
Aerial Parts Chapter I Preliminary Phytochemical Screening
of the Air Dried Powdered Aerial Parts of Anisotes trisulcus
(Forssk.) Nees. The dried aerial parts of Anisotes trisulcus
(Forssk.) Nees. contains volatile substances, carbohydrates
and/or glycosides, unsaturated sterols and/or triterpenes,
tannins, flavonoids, lactones and/or esters in addition to
alkaloids and/or basic nitrogenous substances. Chapter II
Extraction, Fractionation and Isolation of the Constituents
from Anisotes trisulcus (Forssk.) Nees. Aerial Parts The airdried powdered aerial parts (2.5 kg) of Anisotes trisulcus
(Forssk.) Nees.were extracted by maceration and percolation
with (70%) methanol till complete exhaustion [four times each
10 L, overnight]. The combined methanolic extracts were
concentrated under reduced pressure till constant weight to
give a dark green syrupy residue (300 gm). The methanolic
extract (300 gm) was digested in the least amount of distilled
water subjected to successive solvent fractionation using a
separating funnel with n-hexane, chloroform, ethyl-acetate, nbutanol. Each extractive was concentrated separately under
reduced pressure to give 45, 35, 18 and 8 gm respectively.
The lipoidal content of the n-hexane fraction was
investigated. The obtained different extractives were
subjected to different chromatographic techniques for
separations of their constituents where 20 compounds were
isolated. Chapter III Identification of the Isolated Compounds
from Anisotes trisulcus (Forssk.) Nees. Aerial Parts Structure
elucidation of the isolated compounds was deduced on the
basis of spectroscopic methods: (UV, IR, 1H-NMR,1H-1H
COSY, 13C-NMR, DEPT 13C-NMR, HSQC, HMQC, EI,+FAB- and MALDI-TOF-MS) in addition to comparison of the
physical, chemical and chromatographic characters of these
compounds with the available authentic samples. A list of the
identified compounds listed in Table (66). Table (66): List of
the isolated compounds from Anisotes trisulcus (Forssk.)
Nees.aerial parts. Sign Name and Structure Remarks AT-1 αAmyrin First report from the plant AT-2 β-Sitosterol First
report from the plant AT-3 Stigmasterol First report from the
plant Table (66): Continued. Sign Name and Structure
Remarks AT-4 (2S, 3S, 4R)-2[(2’R)-2’-Hydroxytetracosanoyl
amino] octadecane 1,3,4-triol First report from the family
Acanthaceae AT-5 3,7,8,3’-Tetrahydroxy 5-methoxy-4’benzoyl flavone New Compound AT-6 3,6,7,3’,4’Pentahydroxy -5-methoxy flavone First report from the family
Acanthaceae AT-7 Vasicine (Peganine) Previously isolated
from the plant Table (66): Continued. Sign Name and
Structure Remarks AT-8 Vasicinone Previously isolated from
the plant AT-9 Anisotine Previously isolated from the plant
AT-10 5-Hydroxyvasnetine New compound AT-11 Veratric
acid First report from the family Acanthaceae Table (66):
Continued. Sign Name and Structure Remarks AT-12 3Methoxy,4-hydroxy benzoic acid (Vanillic acid) First report
from the genus Anisotes AT-13 β-Sitosterol-3-O-β-Dglucopyranoside First report from the genus Anisotes AT-14
7-Hydroxyvasicine (Vasicinol) First report from the genus
Anisotes AT-15 7-Hydroxyvasicinone (Vasicinolone) First
report from the genus Anisotes AT-16 8-Amino-7,8,9,11tetrahydro-6H-pyrido[2,1-b] quinazoline-2,6-diol New
Compound Table (66): Continued. Sign Name and Structure
Remarks AT-17 8-Amino-3,6-dihydroxy-7,8,9-trihydro-6Hpyrido[2,1-b]quinazoline-11-one New compound AT-18
(Dimethylamino)-N-(hydroxymethyl)-N,Ndimethylmethanaminium chloride New compound AT-19 N[(carboxyamino)methyl]–N,N–dimethyl ethanaminium chloride
New compound AT-20 Choline N-[(carboxyamino)methyl]–
N,N–dimethyl ethanaminium chloride First report from the
plant PART II Phytochemical Study of Blepharis ciliaris (L.)
B.L.Burtt. Aerial Parts Chapter I Preliminary Phytochemical
Screening of the Powdered Blepharis ciliaris (L.)
B.L.Burtt.aerial parts The dried aerial parts of Blepharis
ciliaris (L.)B.L.Burtt. contain volatile substances,
carbohydrates and/or glycosides, unsaturated sterols and/or
triterpenes, tannins, flavonoids in addition to lactones and/or
esters. Chapter II Extraction, Fractionation and Isolation of
the Constituents from Blepharis ciliaris (L.) B.L.Burtt.aerial
parts The air-dried powdered aerial parts (3 kg) of Blepharis
ciliaris (L.) B.L.Burtt.were extracted by maceration and
percolation with methanol till complete exhaustion [four times
each 10 ml, overnight]. The combined methanolic extracts
were concentrated under reduced pressure till constant
weight to give a dark green syrupy residue (270 gm). The
methanolic extract (270 gm) was digested in the least amount
of distilled water subjected to successive solvent fractionation
using a separating funnel with n-hexane, chloroform, ethylacetate, n-butanol. Each extractive was concentrated
separately under reduced pressure to give 50, 37, 45 and 12
gm respectively. The lipoidal content of the n-hexane fraction
was investigated. The obtained different extractive were
subjected to different chromatographic techniques for
separations of their constituents where 16 compounds were
isolated. Chapter III Identification of the Isolated Compounds
from Blepharis ciliaris (L.) B.L.Burtt. Aerial Parts Structure
elucidation of the isolated compounds was deduced on the
basis of spectroscopic methods: (UV, IR, 1H-NMR,13C-NMR,
DEPT 13C-NMR, EI- and +FAB- MS) in addition to
comparison of the physical, chemical and chromatographic
characters of these compounds with available authentic
samples. A list of the identified compounds are listed in Table
(67). Table (67): List of the isolated compounds from
Blepharis ciliaris (L.) B.L.Burtt.aerial parts. Sign Name and
Structure Remarks BC-1 β-Sitosterol First report from the
plant BC-2 Stigmasterol First report from the plant Table (67):
Continued. Sign Name and Structure Remarks BC-3
Stigmasterol tetracosanoate New compound BC-4 (2S, 3S,
4R)-2[(2’R)-2’-(Hydroxyeicosanoyl amino) octadecane-1,3,4triol First report from the family Acanthaceae BC-5 Methyl
veratrate First report from the family Acanthaceae BC-6
Methyl vanillate First report from the genus Blepharis Table
(67): Continued. Sign Name and Structure Remarks BC-7
Protocatechuic acid First report from the genus Blepharis BC8 Apigenin First report from the plant BC-9 β-Sitosterol-3-O-βD-glucopyranoside First report from the plant BC-10
Stigmasterol-3-O-β-D-glucopyranose First report from the
plant BC-11 Apigenin -7-O- β-D-glucopyranoside Previously
isolated from the plant Table (67): Continued. Sign Name and
Structure Remarks BC-12 Apigenin-7-O-(6’’-E-p-coumaroyl-βD-glucopyranoside) First report from the plant BC-13
Genistein-7-O-(6’’-O-E-caffeoyl)-β-D- glucopyranoside New
compound BC-14 Naringenin-7-O-(3’’-acetyl-6’’-E-pcoumaroyl-β-D-glucopyranoside Previously isolated from the
plant Table (67): Continued. Sign Name and Structure
Remarks BC-15 Naringenin-7-O-(6’’-E-p-coumaroyl-β-Dglucopyranoside First report from the plant BC-16 Acteoside
Previously isolated from the plant PART III Biological Studies
of Anisotes trisuclus (Forssk.) Nees. and Blepharis ciliaris
(L.)B.L.Burtt. Aerial Parts 1- Antioxidant Activity: The total
methanolic and the ethyl acetate extracts of Anisotes trisulcus
(Forssk.) Nees. showed the highest antioxidant activity 75%
and 68% at 1 mg concentration while Blepharis ciliaris (L.)
B.L.Burtt. also exhibited high antioxidant activity 89% and
86% at 1 mg concentration respectively for the same extracts.
The obtained results could be attributed to the presence of
phenolic compounds (flavonoids, phenylpropanoids and
phenolic acids). While the pure compounds apigenin,
apigenin-7-O-β-D-glucopyranoside, genistein-7-O-(6’’-O-Ecaffeoyl)-β-D-glucopyranoside and naringenin-7-O-(6’’-E-pcoumaroyl-β-D-glucopyranoside showed moderate activity,
which was in agreement with the conclusion that the
hydroxylation pattern of the flavonoidal compounds is highly
effective against free radicals. 2- Acute Toxicity Study: The
total methanolic extract of Anisotes trisulcus (Forssk.) Nees.
didn’t show any signs of toxicity and mortality up to 5 gm/kg
while that of Blepharis ciliaris (L.) B.L.Burtt. didn’t show any
signs of toxicity and mortality up to 4 gm/kg. 3- Brine-Shrimp
Assay (Cytotoxic Activity): The activity of the total methanolic
extract and the chloroformic extract of the dried aerial parts of
Anisotes trisulcus (Forrsk.) Nees. was strong in the brine
shrimp bioassay 61% and 60% after 24 hrs while the
methanolic, chloroformic and ethyl acetate showed 70%, 96%
and 68% after 48 hrs respectively which may be attributed to
their alkaloidal, steroidal and/or triterpenoidal content while
on the other hand the n-hexane extract had mild activity.
While the different extracts of the dried aerial parts of
Blepharis ciliaris (L.) B.L.Burtt. showed poor to mild activity
after 24 and 48 hrs respectively. 4- Anti-Inflammatory Activity:
It was observed that all the tested extracts showed antiinflammatory activity in carageenin-induced rat hind paw
oedema model when the animals were pretreated with the
different extracts of the aerial parts of both plants (Anisotes
trisulcus (Forssk.) Nees. and Blepharis ciliaris (L.) B.L.Burtt.)
where the total methanol, n-hexane and ethyl acetate extracts
showed significant anti-inflammatory effect, where marked
reduction of the paw edema was observed due to their high
content of sterols and their corresponding glucosides,
flavonoids and phenolic compounds. 5- Anti-Malarial Activity:
The methanolic, n-hexane and chloroform extracts of
Anisotes trisulcus (Forssk.) Nees. dried aerial parts showed
mild anti-malarial activity against the tested Plasmodium
falciparum (D6 clone) relative to chloroquine. While the ethyl
acetae and the total methanolic extract of Blepharis ciliaris
(L.) B.L.Burtt. dried aerial parts showed mild to weak antimalarial activity against the tested P.falciparum (D6 clone)
relative to chloroquine. 6- Anti-Hyperglycaemic Activity: It was
observed that the total methanolic extract of the aerial parts
of Anisotes trisulcus (Forssk.) Nees. and its n-hexane and
ethyl acetate extracts when given concurrently with the
glucose load at a dose of 400 mg/Kg a significant
hypoglycaemic effect was obtained after 1 hr and this
continued for 2 hrs while the chloroform and the n-butanol
extracts showed the least activity. The obtained results
support the use of powdered Anisotes trisulcus (Forssk.)
Nees. as an anti-diabetic in folk medicine. Concerning the
methanolic extract of the dried aerial parts of Blepharis ciliaris
(L.) B.L.Burtt. and its fractions when given concurrently with
glucose load showed a non-significant anti-hyperglycaemic
activity.