Review jurnal internasional bermeditasi

Aktivitas Anti-Inflamasi Dan Analisis Kualitatif Ekstrak Yang Berbeda Dari Maytenus Obscura

(A. Rich.) Cuf. Oleh Kinerja Tinggi Lapisan Tipis Metode Kromatografi

Disusun Oleh :
Nama

: Nabila Fasya

NIM

: G1F014024

Kelas

:B

JURUSAN FARMASI
FAKULTAS ILMU-ILMU KESEHATAN
UNIVERSITAS JENDERAL SOEDIRMAN
2016

1.

LATAR BELAKANG
Standarisasi tanaman bahan yang digunakan
dalam pengobatan tradisional sangat penting dalam

- Diekstrak dengan 90% ethanol (3x3L) pada

suhu ruang
Ekstrak

sistem perawatan kesehatan. Profil herbal farmakope

- Dipekatkan dibawah vakum

perlu diperbarui untuk menyertakan sidik jari dari

Residu lilin hitam 182, 5g
- Diambil 120g
- Dihilangkan lemaknya dengan petroleum eter
(4x200mL)

herbal obat yang dihasilkan oleh teknologi canggih.

Kinerja tinggi kromatografi lapis tipis (HPTLC)
dapat memberikan sidik jari kromatografi obat herbal
yang

dapat

divisualisasikan,

disimpan

Hasil

sebagai

gambar elektronik dan digunakan sebagai referene

untuk pengendalian kualitas herbal. Karena biaya
rendah,

sampel

throughput

yang

tinggi

dan

pembersihan sampel rendah, HPTLC menjadi salah

satu teknik pengujian kualitas standar. Disamping itu
HPTLC memiliki keuntungan dari analisis simultan
jumlah besar sampel, sehingga mengurangi waktu

Sisa ekstrak
- difraksikan dengan CH2CL2
13,2 g SF dan 99,1 AESF
3. 3 Skrining fitokimia
Skrining fitokimia dilakukan dengan
metode Kokate dan Gokhale, dan Khandelwal

dan biaya per analisis

4.4
2.

Rumusan Masalah
1.

Apa kandungan Maytenus obscura

2.

Apakah ada aktivitas inflamasi dalam M. obscura

3.

Apakah

HPTLC

fingerprint

dapat

Profil HPTLC
a. Metode Kromatografi
Sidik jari diklorometana dan
ekstrak ethanol dari M.
obscura

- Dieksekusi oleh bercak 5, 10, 15, 20

(L) dari sampel SF dan AESF pada
plate HPTLC
- Dilakukan
kromatogravi
pada
20cmx20cm glass HPTLC plate
precoated dengan 200 m silika gel
60F254
- Diaplikasikan lebar 6mm dan 8mm
terpisah dengan cara camag

mengidentifikasi kualitas suatu obat bahan alam

3.

TUJUAN PENELITIAN
Tujuan dari penelitian ini adalah untuk mengevaluasi
dan membandingkan aktivitas anti inflamasi dan
untuk mengembangkan HPTLC fingerprint profil dari
alkohol dan diklorometana ekstrak udara bagian dari
M. obscura.

4. METODE PENELITIAN
3. 1. Bahan Tanaman
M. obscura didapatkan dari Abha, Kerajaan
Saudi arabia. Yang telah diidentifikasi oleh Dr.
Mohammad Atiqur Rahman (taksonomi Obat,
Aromatik

dan

Penelitian,

Beracun

Perguruan

Tanaman
Tinggi

Pusat

Hasil
b.

Pengembangan Sistem Pelarut

Nomor system pelarut telah diuji,
tetapi resolusi terbaik yang diperoleh
dalam toluena pelarut: ethyacetate : asam
asetat glasial (5: 3: 0,2).

Farmasi,

Universitas Raja Saud, Saudi Arabia).

c. Deteksi bintik

3. 2. Persiapan Dan Ekstraksi Bahan Tanaman

Plate

M. Obscura 1,3g

- Diaktifkan dan dikeringkan pada

o
100 C dalam oven selama 3 menit

- Disimpan
di
ruang
fotodokumentasi (Camag )
- Reprostar 3) dan foto yang diambil
dibawah lampu UV 254nm dan
366nm
Nilai Rf
- Direkam oleh perangkat lunak WIN
CATS
Hasil
d.

Aktivitas Anti-Inflamasi
Tikus wistar jantan (100-150g)

Kontrol

Standart

- Diberikan 0,1
mL 10%
formalin cair

Diinjeksikan (i.p.)
Indometasin (30mg
/kg)

Edema

- Diinjeksikan anti-inflamasi dari

SF dan AESF (100, 200mglKg)
- Dicatat setelah 60 menit
menggunakan hidro plethys
mograph
- Dibandingkan
- Dikuantifikasikan
dengan
rumus
Hasil

Rumus :
Perubahan volume edema = (L2-R2) - (L1-R1) di mana L1
adalah Volume kaki kiri sebelum injeksi formalin; R1 adalah
volume yang kaki kanan sebelum menyuntikkan kaki kiri
dengan formalin; L2 yang tersisa kaki Volume 1h setelah
suntik formalin dan 2h setelah pengobatan; R2 adalah volume
cakar 1h setelah injeksi formalin di kaki kiri dan 2h setelah
perawatan.
e. Analisis statistic
Hasil dianalisis menggunakan salah satu cara ANOVA
dan Dunnett tes sebagai pos hoctest (lebih dari
2groups) atau Kruskal Tes wallis ANOVA dengan
Dunn sebagai pos hoctest (kecuali dinyatakan lain)
dan disajikan sebagai mean SEM. hanya hasil
dengan P0.05 dianggap sebagai signifikan [20].

5.

Hasil dan Pembahasan

4.1. Skrining Fitokimia Dari Aesfand SF
Skrining fitokimia Apreliminary dari AESF
and SF menunjukkan bahwa kedua ekstrak
mengandung protein, lipid, karbohidrat,
menurunkan gula, fenol, tanin, flavonoid,
saponin, triterpenoid, alkaloid dan kuinon
(Tabel 1).
4.2. HPTLC fingerprinting dari AESF and SF
Tabel 2 dan 3 menunjukkan bahwa HPTLC
fingerprinting dari AESF and SF, masing-masing
menimbulkan beberapa puncak. Profile HPTLC
dari AESF and SF dibawah UV 366 and 254nm
telah dicatat masing-masing dalam Angka 1
dan 2.(figure 1). Kromatogram HPLC yang
sesuai disajikan dalam Angka 3A-3H (figure 2)
The ACES menunjukkan 6, 7, 8 dan 8 spot
dengan nilai Rf dalam kisaran 0.28 sampai 0.80
untuk 5, 10, 15 dan 20 L (Tabel 2dan Angka
3A-3D). Namun, SF menunjukkan 10, 11, 9 dan
8 spots dengan nilai Rf kisaran dari 0.12 sampai
0.76 untuk 5, 10, 15dan 20 L (Tabel 3and
Angka 3E-3H).

4.3. aktivitas anti-inflamasi AESF dan SF

Injeksi formalin ke dalam tikus kaki
belakang mengakibatkan pembengkakan sedini
10 menit setelah injeksi. pembengkakan ini
mencapai dataran tinggi dengan 30 menit dan
dipertahankan hingga 240min [21]. Pengobatan
tikus (i.p.) dengan AESF dan SF dari dosis M.
obscurain 100 dan 200mg/kg menghambat
peradangan formalin yang diinduksi oleh 50%
dan 45,5%, 55,9% dan 51,4%, (Tabel 4)

6. KESIMPULAN
Aktivitas anti-inflamasi tergantung dosis
dari kedua ekstrak dapat menjelaskan setidaknya
sebagian
alasan
di
luar
menggunakan
Maytenusspecies dalam pengobatan inflamasi
penyakit [1]. Skrining fitokimia dari kedua ekstrak
menunjukkan adanya flavonoid yang dikenal
memiliki fungsi anti-inflamasi dan antioksidan
kegiatan [22]. Kedua ekstrak memiliki kandungan
flavonoid hamper sama dan memiliki presentase
penutupan penghambatan pada peradangan. HPTLC
fingerprinting terbukti menjadi cara tepat, metode
yang akurat untuk identifikasi herbal dan dapat
digunakan lebih lanjut dalam otentikasi dan
standarisasi tanaman penting medicinally.

152

Asian Pac J Trop Biomed 2014; 4(2): 152-157

Contents lists available at ScienceDirect

Asian Pacific Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Document heading

doi:10.1016/S2221-1691(14)60224-0

2014

by the Asian Pacific Journal of Tropical Biomedicine. All rights reserved.

Anti-inflammatory activity and qualitative analysis of different extracts

of Maytenus obscura (A. Rich.) Cuf. by high performance thin layer

chromatography method

Mohamed F. Alajmi , Perwez Alam

*

Deptartment of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia

PEER REVIEW

ABSTRACT

Peer reviewer
Dr. Prawez Alam, Assistant Professor,
Salman bin Abdul Aziz University, Al
Kharj, Kingdom of Saudia Arabia.
Tel: +96615886014
Fax: +96615886001
E-mail: prawez007@gmail.com

Objective: To perform aqueous ethanol soluble fraction (AESF) and dichloromethane extract of
aerial parts of Maytenus obscura (A. Rich.) Cuf. using high performance thin layer chromatography
(HPTLC) and to test anti-inflammatory activity of these extracts.
Methods: HPTLC studies were carried out using CAMAG HPTLC system equipped with Linomat
IV applicator, TLC scanner 3, Reprostar 3, CAMAG ADC 2 and WIN CATS-4 software were used. The
anti-inflammatory activity was tested by injecting different groups of rats (6 each) with formalin
in hind paw and measuring the edema volume before and 1 h later formalin injection. Control
group received saline i.p. The extracts treatment was injected i.p. in doses of 100 and 200 mg/kg 1
h before formalin administration. Indomethacin (30 mg/kg) was used as standard.
Results: The results of preliminary phytochemical studies confirmed the presence of protein,
lipid, carbohydrate, phenol, flavonoid, saponin, triterpenoid, alkaloid and anthraquinone in both
extracts. Chromatography was performed on glass-backed silica gel 60 F254 HPTLC plates with
the green solvents toluene: ethyacetate: glacial acetic acid (5:3:0.2, v/v/v) as mobile phase. HPTLC
finger printing of AESF revealed major eight peaks with Rf values in the range of 0.28 to 0.80 and
the dichloromethane revealed major 11 peaks with Rf values in the range of 0.12 to 0.76. The purity
of sample was confirmed by comparing the absorption spectra at start, middle and end position
of the band. Treatment of rats (i.p.) with AESF and dichloromethane in doses of 100 and 200 mg/kg
inhibited singnificantly (P<0.05, n=6) formalin-induced inflammation by 50%, 55.9%, 45.5%, and
51.4%, respectively.
Conclusions: HPTLC finger printing of AESF and dichloromethane of Maytenus obscura revealed
eight major spots for alcoholic extracts and nine major spots for dichloromethane extracts.
These HPTLC profiles may be of great usefulness in the quality control of herbal products
containing these extracts. The anti-inflammatory activity of both extracts also revealed the
medicinal importance of these extracts. The plant can be further explored for the isolation of
phytoconstituents having anti-inflammatory activity.

Comments
T he research represents advanced

methodological qualitative analysis

of complex matrix such as herbal
medicine where only very limited
information can be acquitted
using traditional techniques. T he
manuscript is well written. I t has
been organized according to well
documented researches. The materials
and methods are written precisely
and well referenced. The results are
compatible with what the methods may
generate. The results were discussed
scientifically according to the results
obtained.
Details on Page 156

KEYWORDS
Maytenus obscura, Phytochemical screening, Finger print, Anti-inflammatory, HPTLC

1. Introduction
Maytenus obscura (A. Rich.) Cuf. (M. obscura) belongs
to the genus Maytenus which is distributed worldwide,
particularly in subtropical and tropical regions, Africa,
*Corresponding author: Mohamed F. Alajmi, Department of Pharmacognosy, College
of Pharmacy, King Saud University, Riyadh, Kingdom of Saudi Arabia.
Tel: +966505151846
E-mail: alajmister@gmail.com
Foundation Project: Supported by College of Pharmacy and the deanship of Scientific
Research of the King Saud University (Riyadh, Saudi Arabia) (Grant No. RGP-VPP-150).

China, Brazil, Paraguay, Uruguay and Argentina and in

southern regions of Saudi Arabia[1]. The genus Maytenus

is rich in triterpenes, diterpenes, sesquiterpene alkaloids

and spermidine alkaloids[2]. Other secondary metabolites
isolate from different species of the genus Maytenus,
Article history:
Received 25 Dec 2013
Received in revised form 1 Jan, 2nd revised form 6 Jan, 3rd revised form 10 Jan 2014
Accepted 16 Jan 2014
Available online 28 Feb 2014

Mohamed F. Alajmi and Perwez Alam /Asian Pac J Trop Biomed 2014; 4(2): 152-157

including flavonoids and flavonoid glycosides[3], phenolic

glucoside and agarofurans[4]. Species belonging to this genus
are extensively investigated for bioactive compounds as
they are widely used in folk medicine such as antiseptic,
antiasthmatic, fertility-regulating agents, antitumor and
antiulcer[5,6]. S ome Maytenus species are marketed in
the form of capsules, powders, dried leaves, fresh leaves,
aqueous or aqueous-alcoholic preparations[5]. Herbal drugs
play major role in treatment of many diseases especially
in undeveloped countries [7] . S tandardization of plant
materials used in traditional medicine is very important in
todays health care system. Pharmacopoeial herbal profiles
need to be updated to include the fingerprinting of herbal
drugs generated by advanced technology. These modern
methods describing the identification and quantification of
active constituents in the plant material may be useful for
complete standardization of herbals and its formulations[8].
High performance thin layer chromatography (HPTLC) can
provide chromatographic fingerprints of herbal drugs that
can be visualized, stored as electronic images and used as
referene for quality control of herbs. Due to its low cost,
high sample throughput and low sample cleanup, HPTLC may
become one of standard quality testing techniques. HPTLC
has the advantage of simultaneous analysis of large numbers
of samples, thus reducing time and cost per analysis[9-13].
The aim of this study is to evaluate and compare the antiinflammatory activity and to develop HPTLC fingerprint
profile of alcoholic and dichloromethane extract of aerial
parts of M. obscura.
2. Materials and methods
2.1. Plant material
M. obscura was collected from Abha, kingdom of Saudi
A rabia. T he plant was collected and identified by D r.
M ohammad A tiqur R ahman ( taxonomist of M edicinal,
Aromatic and Poisonous Plants Research Center, College
of Pharmacy, King Saud University, Saudi Arabia). Voucher

specimens ( 14295 , 13420 and 14309 respectively ) were

deposited in the herbarium of the College of Pharmacy, King
Saud University, Saudi Arabia. Aerial parts were shade dried
and coarsely powdered.
2.2. Preparation and extraction of plant material
About 1.3 kg of M. obscura was exhaustively extracted
at room temperature with 90% ethanol (33 L). The ethanol
extract was then concentrated under vacuum to yield 182.5
g black waxy residue. A total of 120 g was defatted with
petroleum ether (4200 mL). The remaining alcoholic extract
was fractionated with CH2Cl2 (5200 mL) to yield 13.2 g CH2Cl2

soluble fraction
fraction (AESF).

(SF)

153

and 99.1 g aqueous ethanol soluble

2.3. Phytochemical screening

Preliminary phytochemical screening was done following
the method of Kokate and Gokhale[14], and Khandelwal[15].

2.4. HPTLC profile

2.4.1. Chromatographic method
HPTLC studies were carried out following the method
of H arborne and W agner et al [16,17] . T he protocol for
preparing sample solutions was optimized for high quality
fingerprinting. The fingerprinting of dichloromethane and
alcoholic extracts of M. obscura were executed by spotting
5, 10, 15, 20 L of suitably diluted sample solution of the SF
and AESF on a HPTLC plate. Chromatography was performed
on 20 cm10 cm glass HPTLC plates precoated with 200 m
layers of silica gel 60F254 (E. Merck, Darmstadt, Germany).
Samples (5 L, 10 L, 15 L, 20 L) of each extract were
applied as bands 6 mm wide and 8 mm apart by means of
Camag (Muttenz, Switzerland) Linomat IV sample applicator
equipped with a 25 L syringe which was programmed
through WIN CATS software. The developed plate was dried at
100 C in hot air oven for 3 min. The plate was kept in photodocumentation chamber (CAMAG Reprostar 3) and captured
the images under UV light at 254 and 366 nm, respectively.
The Rf values and finger print data were recorded by WIN
CATS software.
2.4.2. Solvent system development
A number of solvent systems were tried, but the best
resolution was obtained in the solvent toluene: ethyacetate:
glacial acetic acid (5:3:0.2).
2.4.3. Detection of spots
The developed plate was dried at 100 C in hot air oven for
3 min to evaporate solvents from the plate. The plate was
kept in photo-documentation chamber (CAMAG Reprostar
3) and images captured under UV light at 254 and 366 nm,
respectively. T he R f values and finger print data were
recorded by WIN CATS software.
2.5. Anti-inflammatory activity
Inflammation was induced in the plantar side of the

left hind paws of male wistar rats (weighing 100-150 g) by

injecting 0.1 mL of 10% aqueous formalin as described by Fu
et al[18]. The increase in the paw edema was recorded prior
to and 60 min after formalin injection (or 2 h after treatment
with extracts) using hydro-plethysmograph (Model 7150,
Ugo, Basile, Haly). Instead of the treatment (extracts) (in the

154

Mohamed F. Alajmi and Perwez Alam /Asian Pac J Trop Biomed 2014; 4(2): 152-157

treated group), the control group was administered (i.p.) 0.1

mL of 10% aqueous formalin and the standard group was
given (i.p.) injection of Indomethacin (30 mg/kg)[19]. Treated
group: the anti-inflammatory activity of the SF and AESF (100
and 200 mg/kg) were tested by injecting the treatment (SF
and AESF) solubilized in suitable vehicle as described above
60 min (i.p.) before formalin injection. Then the procedure
was continued as outlined above. Effect of the SF and AESF
on the formalin-induced paw volume was recorded (2 h
after extract treatment or 1 h after formalin injection) as
percentage inhibition relative to the control untreated group
and were compared to the standard group. Change in volume
of edema was quantified using the following formula:
Change in volume of edema=(L2-R2)-(L1-R1)

Where L1 is left paw volume before injection of formalin; R1

is right paw volume before injecting left paw with formalin;

L2 is left paw volume 1 h after injecting formalin and 2 h after
treatment; R2 is right paw volume 1 h after formalin injection
in the left paw and 2 h after treatment.

Table 2
HPTLC profile of the AESF.
Volume of AESF applied

Peak
3

0.51

0.35

5 L

0.77

2139.4

0.35

1367.2

0.50

1889.4

0.41

0.62

0.76

1524.7

0.34

1868.5

0.50

2505.9

0.70

1084.5

0.80

877.2

0.70

0.29

0.41

0.62

0.74

0.28

4
6

0.74

0.69

5 L

3. Results

3.1. Phytochemical screening of AESF and SF

Table 1
Phytochemical contents of AESF and SF.

AESF
+

++

++

++

+++

+++

+++

+++

++
++
++

3.2 HPTLC fingerprinting of AESF and SF

++

Tables 2 and 3 show HPTLC fingerprinting of AESF and SF,

respectively, which revealed several peaks.

1062.5

0.50
0.61

0.69
0.12
0.20

195.5
149.0

2549.7
6260.4
1743.5

10379.4

1863.2
596.7

1342.3
109.1
424.0

1312.0
9381.1

0.50

16303.8

10

0.69

823.9

0.16

0.23
0.41
0.61

0.75

4143.7
299.3

3205.5
3360.9
796.6
379.9

0.20

1574.0

0.33

10890.1

0.49

20910.9

0.69

848.7

0.20

1950.2

0.33

11055.3

0.49

17840.5

0.69

5
7

2
3
20 L

933.0

2222.8

0.33

1103.6

++

689.0

0.28

15 L

997.2

11

0.34

0.41

0.16

0.29

SF

+
+

10 L

1548.8

262.6

0.76

638.1

0.19

10
1

A preliminary phytochemical screening of AESF and

600.0

Height area

0.23

837.4

Rf

showed that both extracts contain protein, lipid,

carbohydrate, reducing sugar, phenol, tannin, flavonoid,
saponin, triterpenoid, alkaloid and quinone (Table 1).

0.80

0.16

961.4

3439.8

0.61

503.1

0.49

Peak

452.0

2358.8

0.40

599.3

0.33

Volume of SF applied

772.6

0.29

Table 3
HPTLC profile of the SF.

247.8

20 L

810.1

1638.9

0.73

15 L

337.3

0.63

10 L

Height area

Results were analyzed using one way ANOVA and Dunnett

test as post hoc test (for more than 2 groups) or Kruskals
wallis ANOVA with D unns test as post hoc test ( unless
otherwise stated) and presented as meanSEM. Only results
with P0.05 were regarded as significant[20].

Chemical constituents
Protein
Lipid
Carbohydrate
Phenol
Flavonoid
Saponin
Triterpenoid
Alkaloid
Anthraquinone

0.30

2.6. Statistical analysis

SF

Rf

4
6

0.28
0.40
0.60

0.74

0.28
0.40
0.60

0.75

5098.2
4536.3
4063.2
958.3

6013.3
9566.2
4102.5
769.3

1373.3

155

Mohamed F. Alajmi and Perwez Alam /Asian Pac J Trop Biomed 2014; 4(2): 152-157

profile of AESF and SF under UV 366 and 254 nm is

recorded in the Figures 1 and 2, respectively.

The corresponding HPTLC chromatograms are presented in

Figures 3A-3H.

HPTLC

The AESF revealed 6, 7, 8 and 8 spots with R f values in

the range of 0.28 to 0.80 for 5, 10, 15 and 20 L application
volume, respectively (Table 2 and Figures 3A-3D). However,

Figure 1. HPTLC picture of the AESF and SF of sample application at UV 254

nm.
A: AESF at 5, 10, 15 and 20 L; B: SF at 5, 10, 15 and 20 L.
1000

AU

Track 1 ID: EtOH Extract of MO

1000

900

AU

600

600
500

400

400

300

300

200

100

AU

0.00

0.20

0.60

Track 4 ID: EtOH Extract of MO

1.00

Rf

600
500
400
300
1
0.00

0.20

0.40

200

7
0.80

1.00

1000

600

200

100
0.00

AU

0.20
1000

300

0.40

6 7

200

0.60

1.00

Track 7 ID: EtOH Extract of MO

Rf

0.00

0.20

900

AU

700
600

0.40
1000
900

200

10

0.60

12

100

0.80

1.00

Rf

0.00

0.20

0.40

0.60

Track 8 ID: EtOH Extract of MO

H

500
400

300
200

100
0.00

600

0.40

400
7

0.60

300
8

200

500

0.20

700

4
6

100
0.80

0.80

1.00

Rf

800

800

400

300

1 2 3

100

0.80

0.60

7 8

500

400

300

800

600

500

0.40

Track 6 ID: EtOH Extract of MO

700

500

400

0.20

900

700

600

0.00

AU

800

800

Rf

900

700

100

0.60

Track 5 ID: EtOH Extract of MO

1000
E
AU

900

800

100

0.80

Track 3 ID: EtOH Extract of MO

700

200

5 6

0.40

900

AU

700

500

1000

1000

800

800

Track 2 ID: EtOH Extract of MO

900

700

Figure 2. HPTLC picture of the AESF and SF of sample application at 366 nm.
C: AESF at 5, 10, 15 and 20 L; D: SF at 5, 10, 15 and 20 L.

1.00

Rf

0.00

0.20

0.40

0.60

7 8

0.80

1.00

Rf

Figure 3. HPTLC chromatogram of AESF and SF showing different peaks of phytoconstituents.

A: AESF (5 L); B: AESF (10 L); C: AESF (15 L); D: AESF (20 L); E: SF (5 L); F: SF (10 L); G: SF (15 L); H: SF (20 L). MO: M. obscura.

10 11
0.80

1.00

Rf

156

Mohamed F. Alajmi and Perwez Alam /Asian Pac J Trop Biomed 2014; 4(2): 152-157

revealed 10, 11, 9 and 8 spots with Rf values in the range

of 0.12 to 0.76 for 5, 10, 15 and 20 L application volume,
respectively (Table 3 and Figures 3E-3H). The purity of the
sample was confirmed by comparing the absorption spectra
at start, middle and end position of the band.
SF

3.3. Anti-inflammatory activity of AESF and SF

Injection of formalin into a rats hind paw resulted in
swelling as early as 10 min after injection. This swelling
reached a plateau by 30 min and was maintained for up to
240 min[21]. Treatment of rats (i.p.) with AESF and SF of M.
obscura in doses of 100 and 200 mg/kg inhibited formalininduced inflammation by 50% and 45.5%, 55.9% and 51.4%,
respectively (Table 4).
Table 4
Effect of AESF and SF on formalin-induced inflammation in rats.
Treatment
Control
AESF
SF

Conflict of interest statement

We declare that we have no conflict of interest.

Dose (mg/kg)

Paw edema
volume (mL)

Inhibition (%)

100

0.340.04

50.0

Acknowledgements

0.370.02

45.5

The authors are grateful to the research center in College

of Pharmacy and the deanship of Scientific Research of the
King Saud University (Riyadh, Saudi Arabia) for supporting
this project (Grant No. RGP-VPP-150).

0.1 mL (formalin)

0.680.06

200

0.300.01

200

0.330.01

100

Indomethacin

of activity according to the percentage content of these UV

active compounds to the present extracts content. This
might form a target for more comprehensive studies. The
presence of any adulterant will be easily traced having
these profiles developed and stored in a library. Literature
review revealed that there is no previous work that has
been done on these two extracts. AESF and SF of M. obscura
produced dose-dependent significant anti-inflammatory
activity in rats. This activity may be partially explained
in view of the high content of flavonoids in both extracts.
HPTLC profiles of both extrats revealed the presence of eight
to nine major spots absorbing in different UV wavelengths,
pointing to the difference in these compounds.

30

**
*

**

0.100.02

**

0.0

55.9
51.4
85.2

4. Discussion
The dose-dependent anti-inflammatory activity of both

extracts may explain at least partly the rationale beyond

using the Maytenus species in treatment of inflammatory
diseases [1] . P hytochmical screening of both extracts
revealed the presence of flavonoids which are known to
possess anti-inflammatory and antioxidant activities[22].
Flavonoid content of both extracts are almost equal and
hence both extracts have close percentage of inhibition on
inflammation. However, it is evident that they are different
in polarity and UV absorption. Therefore, the presence of
these compounds might be the ultimate cause for their
bioactivity. HPTLC analysis of AESF and SF extracts revealed
different peaks which are distinct for each extract and
are concentration-dependent. Therefore, any additive or
coamixed material will definitely affect the HPTLC picture
showing new unrelated peaks pointing to the presence of
either adulteration or deterioration. HPTLC fingerprinting
is proved to be a liner, precise, accurate method for herbal
identification and can be used further in authentication and
standardization of the medicinally important plant. Such
finger printing is useful in quality control of herbal products
and checking for the adulterant. Therefore, it can be useful
for the evaluation of different marketed pharmaceutical
preparations and plant systematic studies[13].
Hence, HPTLC provides great utility for the quality control
of these products. It may be utilized to estimate the extent

Comments
Background
Many countries (including developed ones) suffer a big
problem in standardizing and quality control of the herbal
plants. This is due to many factors among which are the
complex form of these products and the inability of the
traditional methods to precisely estimate the quality of
the herbs. Regulatory authorities used to follow tedious
procedures that may give indication about the quality of the
herb but not complete map of the chemical content (which
is vital for the biological activity of these plants).
Research frontiers
The research represents an advancement in the quality
control of the herbal medicine. It provides new tool for
fingerprinting using precise chemical contents mapping
which might be utilized in correct identification of not only
identity of the plant but even the purity of the plant product
and the absence of contaminants. The authors utilized one
of the advanced technologies i.e. HPTLC for that purposes
which affords fast, reliable and precise tool for this purpose.
Related reports
No similar reports were found in the literature regarding
this plant specifically, since it is being evaluated using
this tool for the first time as per literature review available

Mohamed F. Alajmi and Perwez Alam /Asian Pac J Trop Biomed 2014; 4(2): 152-157

in our resources.
Innovations and breakthroughs
The previous pharmacopeial methods for testing quality
control concentrate on organoleptics, ash, and vague
chemical screening which could not give precise chemical
contents. Chemical content is the driving force for the
medicinal value of the plant. This is why using such new
techniques assure not only identity, purity of the plant but
even the activity against the claimed diseases.
Applications
It can help to sensitize other groups of researches and
adapt similar methodology in quality studies of other plants.
It can be utilized even for microbial fermentation products
and the presence of the analyte of interest. It may play a
good role in food, food supplement screening and quality
control.
Peer review
T he research represents advanced methodological
qualitative analysis of complex matrix such as herbal
medicine where only very limited information can be
acquitted using traditional techniques. The manuscript
is well written. It has been organized according to well
documented researches. The materials and methods are
written precisely and well referenced. T he results are
compatible with what the methods may generate. T he
results were discussed scientifically according to the results
obtained.
References
[1] Rojo de Almeida MT, Rios-Luci C, Pardn JM, Palermo JA.
Antiproliferative terpenoids and alkaloids from the roots of
Mayenus vitis-idaea and Maytenus spinosa. Phytochemistry 2010;
71(14-15): 1741-1748.
[2] Gutierrez F, Estvez-Braun A, Ravelo AG, Astudillo L, Zrate R.
Terpenoids from the medicinal plant Maytenus ilicifolia. J Nat
Prod 2007; 70(6): 1049-1052.
[3] Momtaz S, Hussein AA, Ostad SN, Abbdollahi M, Lall N. Growth
inhibition and induction of apoptosis in human cancerous Hela
cells by Maytenus procumbens. Food Chem Toxicol 2013; 51: 3845.
[4] Crestani S, Rattmann YD, Cipriani TR, de Souza LM, Lacomini
M, Kassuya CA, et al. A potent and nitiric oxide-dependent
hypotensive effect induced in rats by semi-purified fractions
from Maytenus ilicifolia. Vascul Pharmacol 2009; 51(1): 57-63.
[5] C osta PM , F erreira PM , B olzani VS , F urlan M , de F reitas
F ormenton M acedo D os S antos V A , C orsino J , et al.
Antiproliferative activity of pristimerin isolated from Maytenus
ilicifolia (Celastraceae) in human HL-60 cells. Toxicol In Vitro
2008; 22(4): 854-863.
[6] B ramhachari PV , R avichand J , R eddy YH , K otresha D ,

157

Viswanatha Chaitanya K, Bobbarala V. Evaluation of hydroxyl

radical scavenging activity and HPTLC fingerprint profiling of
Aegle marmelos (L.) Correa extracts. J Pharm Res 2011; 4(1): 252.
[7] K aushik S , S harma P , J ain A , S ikarwar MS . P reliminary
phytochemical screening and HPTLC fingerprinting of Nicotiana
tabacum leaf. J Pharm Res 2010; 3(5): 144.
[8] K aul N , A grawal H , P atil B , K akad A , D haneshwar SR .
Application of stability-indicating HPTLC method for quantitative
determination of metadoxine in pharmaceutical dosage form.
Farmaco 2005; 60(4): 351-360.
[9] Faiyazuddin Md, Baboota S, Ali J, Ahuja A, Ahmad S, Akhtar
J. Validated HPTLC method for simultaneous quantitation of
bioactive citral isomers in lemongrass oil encapsulated solid lipid
nanoparticle formulation. Int J Essen Oil Ther 2009; 3: 142-146.
[10] Faiyazuddin M, Ali J, Ahmad S, Ahmad N, Akhtar J, Baboota S.
Chromatographic analysis of trans and cis-citral in lemongrass
oil and in a topical phytonanocosmeceutical formulation, and
validation of the method. J Planar Chromat 2010; 23(3): 233-236.
[11] F aiyazuddin M d, A hmad S , I qbal Z , T alegaonkar S , A hmad
FJ , B hatnagar A , et al. S tability indicating HPTLC method
for determination of terbutaline sulfate in bulk and from
submicronized dry powder inhalers. Anal Sci 2010; 26(4): 467-472.
[12] Faiyazuddin M, Rauf A, Ahmad N, Ahmad S, Iqbal Z, Talegaonkar
S, et al. A validated HPTLC method for determination of terbutaline
sulfate in biological samples: application to pharmacokinetic
study. Saudi Pharm J 2011; 19(3): 185-191.
[13] Yamunadevi M, Wesely EG, Johnson M. Chromatographic finger
print analysis of steroids in Aerva lanata L. by HPTLC technique.
Asian Pac J Trop Biomed 2011; 1(6): 428-433.
[14] Kokate CK, Gokhale SB. Practical pharmacognosy. Pune: Nirali
Prakashan; 2008, p. 42-44.
[15] Khandelwal KR. Practical pharmacognosy. Pune: Pragati Books
Pvt. Ltd.; 2008, p. 149-160.
[16] Harborne JB. Phytochemical methods. 3rd ed. London: Chapman
and Hall; 1998, p. 44-46.
[17] Wagner H, Baldt S, Zgainski EM. Plant drug analysis. Berlin:
Springer; 1996, p. 85.
[18] Fu KY, Light AR, Maixner W. Long-lasting inflammation and
long-term hyperalgesia anfter subcutaneous formaline injection
into the rat hind paw. J Pain 2001; 2(1): 2-11.
[19] Tavares TG, Spindola H, Longato G, Pintado ME, Caralho JE,
M alcata FX . A ntinociceptive and anti-inflammatory effects
of novel dietary protein hydrolysate produced from whey by
proteases of Cynara cardunculus. Int Dairy J 2013; 32(2): 156162.
[20] D aniel WW . Biostatics: a foundation for analysis in health
sciences. 9th ed. New York: Wiley; 2009.
[21] Cannon KE, Leurs R, Hough LB. Activation of peripheral and
spinal histamine H 3 receptors inhibits formalin-induced
inflammation and nociception, respectively. Pharmacol
Biochem Behav 2007; 88(1): 122-129.
[22] Mendoza-Wilson AM, Glossman-Mitnik D. Theoretical study of
the molecular properties and chemical reactivity of (+)-catechin
and (-)-epicatechin related to their antioxidant ability. J Mol
Struct 2006; 761(1-3): 97-106.