M Salacia reticulata

A
S
T
E
R

D
O
C This Master Document is exclusive intellectual property of
Natural Remedies Pvt. Ltd. This information shall not be revealed
U to any one in part or full without written permission from
Natural Remedies Pvt. Ltd.

M
Developed by

E Quality Control Department

N NATURAL
P R I V A T E
REMEDIES
L I
Plot #5B, Veerasandra Indl. Area, 19th K.M. Stone,
M I T E D

T
Hosur Road, Bangalore - 561 229
Ph: +91-080-7832265, 7833092, 7834872, Fax: 7834369,
E-mail : indherbs@vsnl.com
 Salacia reticulata

CONTENTS
1) Brief literature review on Salacia reticulata (Pg.1-4)
a) Classification
b) Botanical synonym
c) Vernacular names
d) Part used
e) Botanical description
f) Geographical distribution
g) Traditional use
h) Pharmacology
i) Toxicity
j) Phytochemistry
k) Marker compound
l) Active Principles

2) Analytical specifications of Crude Drug (Pg.5-6)

b) Macroscopic characterization
c) Identification of Crude drug by TLC / HPTLC

3) Analytical specifications for Salacia reticulata extract (Pg.7-12)

a) Identification tests
b) Identification of extract by TLC
c) Quantitative determination of Mangiferin in Salacia reticulata by HPLC method
d) Biological standardization

4) Analytical specifications for Mangiferin (Pg. 13-18)

a) UV Spectrum
b) TLC Profile
c) IR Spectrum
1
d) HNMR Spectrum
13
e) C NMR Spectrum
f) Mass Spectrum
g) HPLC Chromatograms

5) References (Pg. 19-21)

1$785$/ 5(0(',(6 ² 5(6($5&+ &(175(

 Salacia reticulata

6alacia reticulata Wight.

(a) Classification:
Kingdom : Plantae
Division : Angiospermae
Class : Dicotyledoneae
Order : Sapindales
Family : Hippocrateaceae
Genus : Salacia
Species : reticulata Wight

(b) Botanical synonym: None Fig. 1

(c) Vernacular name:

English : Salacia
Kannada : Ekanayakam
Malayalam : Ponkoranti, Koranti
Tamil : Ponkoranti
Sanskrit : Vairi, pitika
Telugu : Anukudu cettu
Sinhalese : Himbutu, Kothalahimbutu

(d) Part used: Root

(e) Botanical description: A large, straggling, woody shrub with dichotomous branching. Bark
smooth, greenish grey, thin, white inside. Leaves opposite, elliptic-oblong, base acute, apex
abruptly acuminate, margin toothed with minute rounded teeth, leathery, hairless, shiny, lateral
nerves about seven pairs, prominent beneath; Flowers bisexual, 2-8 clustered in leaf axils,
greenish white to greenish yellow, calyx lobes entire, anthers dehiscing transversely. Drupes
globose, tubercular, pinkish orange when ripe. Seeds 1-4, almond like1-3.

(f) Geographical distribution: Southern India and Sri Lanka. Rare in evergreen forests of western
ghats1-3.

(g) Traditional uses: The roots are acrid, bitter, thermogenic, urinary, astringent, anodyne, anti-
inflammatory. They are useful in vitiated conditions of vata, diabetes, haemorrhoids,
rheumatism, gonorrhoea and skin diseases4-6.

(h) Pharmacology: Serasinghe and coworked orally administered an aqueous extract of Salacia
reticulata prepared from the root bark to streptozotocin-induced diabetic rats. Before drug
administration, blood samples were collected from rats fasted for 16 hours. Subsequently,
selected rats either received water (control group) or different doses of Salacia reticulata
extract. Blood samples were collected every hour for 5 hours to determine the change in blood
plasma glucose levels of treated and control rats. Reductions in plasma glucose levels of more
than 100 mg/dl were observed in 42.8%, 45.4%, and 87.5% of treated rats that received 0.5
g/kg, 1.0 g/kg, and 5.0 g/kg of the Salacia reticulata extract, respectively. The effects of the
extract lasted for the 5-hour duration of the experiment. The blood glucose lowering effects of
Salacia reticulata was persistent7.
NATURAL REMEDIES-RESEARCH CENTRE 1 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]
 Salacia reticulata

Mangiferin, salacinol and kotalanol have been reported to be potent alpha-glucosidase inhibitors
that have been shown to inhibit increases in serum glucose levels8-10. Mangiferin also inhibits
aldose reductase activity, thereby delaying the onset or progression of diabetic complications
(e.g. diabetic neuropathy and nephropathy)9. The polyphenol constituents of S. reticulata, the
catechins, also contribute to the antidiabetic property of the plant9.

Further studies on the inhibitory effects of an aqueous extract from the stem of S. reticulata
Wight (SRE) (Celastraceae) on postprandial hyperglycaemia in rats and humans were
investigated. SRE dose- dependently suppressed elevation of the serum glucose level induced
by sucrose, maltose and alpha-starch, but not by glucose or lactose. The suppressive effect of
SRE was more potent against sucrose-induced hyperglycaemia than against hyperglycaemia
induced by the other sugars. SRE strongly inhibited the activities of alpha-glucosidases prepared
from yeast and rat jejunum (IC50: 5 and 8 micro g/ml, respectively). SRE also inhibited the
activity of alpha-amylase (IC50: 35 micro g/ml), but not that of beta-glucosidase. The relative
inhibitory effects of SRE against partially purified alpha-glucosidases from rat jejunum showed
the order: sucrase=isomaltase (IC50:15 micro g/ml, respectively) > maltase (IC50: 70 micro
g/ml). Moreover, in a sucrose tolerance test performed on human volunteers, 5 min pretreatment
with SRE (200 mg) prior to sucrose (50 g) loading significantly suppressed postprandial
hyperglycaemia. These results demonstrate that, based on its inhibition of alpha-glucosidases
and alpha-amylase, SRE has anti-hyperglycaemic activity, and may be a useful natural material
for the prevention and therapy of diabetes11.

In another study on an animal model (KK-Ay mice) of type 2 diabetes, researchers found that
mangiferin and its glucosides lowered blood glucose levels on oral administration. No effects
were observed in normal mice and beneficial effects on hyperinsulinemia were observed in
diabetic mice. The authors of this study concluded that mangiferin and its glucosides probably
have beneficial effects on blood glucose levels through increasing insulin sensitivity12.

A double-blind placebo-controlled study performed in Japan revealed that blood sugar levels
reduced significantly in humans with mild type 2 diabetes, receiving Salacia reticulata extract
as part of their diet, as compared to control subjects receiving a matching placebo13.
Mangiferin was also found to have beneficial effects in animal models of type 2 diabetes when
coupled with exercise. KK-Ay mice subjected to an exercise regimen and treated with orally
administered mangiferin (30 mg/kg) for two weeks, showed better lipid profiles than control
mice subjected to the exercise regimen and receiving a matching placebo14.

Hyperlipidemic activity: Effects of an aqueous extract from Salacia reticulata (SE), was
investigated for serum lipid levels in rats. Serum triglyceride (TG) in rats given a commercial
diet containing 0.05 or 0.1% SE for 3 Weeks was decreased. SE (0.1%) also suppressed the
increase of serum and liver TG, but slightly increased the serum total and HDL-cholesterol
(Cho) and liver cholesterol in rats given a high sucrose diet for 8 weeks. These results
confirmed that consecutive ingestion of SE lowered the serum TG level, and this activity
appeared to be due to a decrease of absorbed sugars, which are the source of TG in vivo15.

Hepatoprotective and Antioxidant activity: Hot water (SRHW) and methanolic (SRM)
extract of roots and stems methanolic (SRM) extracts from the roots and stems of Salacia
reticulata were examined using an oxidative stress-induced liver injury model. Both SRHW and
SRM extracts (400 mg/kg, p.o.) significantly suppressed the increase in glutamic oxaloacetic
transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in carbon tetrachloride
NATURAL REMEDIES-RESEARCH CENTRE 2 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]
 Salacia reticulata

(CCl4) - treated mice. These extracts also inhibited CCl4-induced thiobarbituric acid-reactive
substance (TBA-RS) formation, which indicates increased lipid peroxidation in the liver.

The IC50 values of the extracts on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

were less than 10 microg/ml and the antioxidative activities of six phenolic compounds from
the roots of S. reticulata were examined. Mangiferin, (-)-4'-O-methylepigallocatechin, and (-)-
epicatechin-(4beta-->8)-(-)-4'-O-methylepigallocatechin, the principal phenolic compounds,
showed potent scavenging activity on DPPH radicals and their concentrations required for 50%
reduction of 40 microM DPPH radicals were 5.9, 10, and 3.2 microM, respectively. On the
other hand, against the CCl4-induced serum GOT and GPT elevations and TBA-RS formation
in mice, mangiferin and (-)-4'-O-methylepigallocatechin showed potent activity at a dose of 100
mg/kg, but (-)-epicatechin-(4beta-->8)-(-)-4'-O-methylepigallocatechin did not. These results
suggest that the antioxidative activity of the principal phenolic compounds is involved in the
hepatoprotective activity of S. reticulata16.

(i) Toxicity study: The safety profile of an extractive from Salacia reticulata stem (SE) was
examined using an oral single dose toxicity test and chromosomal aberration test. In the oral
toxicity test, Sprague-Dawley rats rats were given SE at a dose of 5,000 mg/kg. No deaths or
abnormalities in gross pathological findings were observed during the study period. In the
chromosomal aberration test using cultured mammalian cells (CHL/IU), no cells with
chromosomal aberrations were observed. These results suggest that SE has no serious acute
toxicity or mutagenicity [and can be considered therefore not to prove a hazard during food
preparation]17.

(j) Phytochemistry: Phytochemical studies conducted on Salacia reticulata lead to the isolation
mangiferin, three catechins (-)-epicatechin, (-)-epigallocatechin, and (-)-4’-O-
methylepigallocatechin), two catechin dimers18, epi-kokoondiol19, salacenonal20,
21
salaciquinone , and two novel quinonemethide triterpenoids (celastroloids), isoiguesterinol and
30-hydroxypristimerin23, from the root bark and iguesterin, pristimerin and epi-kokoondiol from
the stem bark22.

Mangiferin (a xanthone from the roots) and sulfonium ion derivatives, kotalanol (from the roots
and stems) and salacinol (from the roots and stems), have been identified as the antidiabetic
principles of S. reticulata through pharmacological studies 8-10.

(k) Marker compound: Mangiferin

HO
O OH
CH2OH
O
HO
OH
HO OH
OH O

C19H18O11 Mol. Wt. 422.35

Mangiferin
Fig. 2

NATURAL REMEDIES-RESEARCH CENTRE 3 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

(l) Active principle:

OH OH

HO
OH

HO OH
HO
O O

SO3
SO 3

HO S+ HO
S+

HO OH
HO OH

C9H18O9S2+ C 12 H 24 O 12 S 2 +
Molecular weight: 334.36 Molecular weight: 424.44
Salacinol Kotalanol
Fig. 3 Fig. 4

All this information provided has been collected from sources considered reliable, but has not been independently
verified by Natural Remedies Pvt. Ltd.

NATURAL REMEDIES-RESEARCH CENTRE 4 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

ANALYTICAL SPE CIFICATION OF THE CRUDE DRUG

Macroscopic Characters:

Colour : Yellowish to golden yellow outside.

Brow to dark brown inside.

Odour : Characteristic odour

Taste : Bitter

Fig. 5

TESTS LIMITS PROTOCOLS

Tests for extraneous material Quality Control Methods
for Medicinal Plant
Materials –WHO
Foreign matter < 1.0% -do-
Sand & Silica Absent -do-
Insect infestation Nil -do-
Rodent contamination Nil -do-
Physico-chemical analysis

Ash content < 15.0%w/w -do-

Acid insoluble ash < 5.0%w/w -do-
Moisture content < 8.0%w/w -do-
(By Loss on drying at 105oC)
Alcohol soluble extractive value Not less than 5% w/w -do-
Water soluble extractive value Not less than 10% w/w
Phytochemical analysis

Mangiferin Positive By HPLC

NATURAL REMEDIES-RESEARCH CENTRE 5 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

IDENTIFICATION OF CRUDE DRUG BY TLC / HPTLC

Sample detail : Salacia reticulata ( Root)

Adsorbent : Precoated silicagel (Al – Sheet)

Solvent system : Ethyl acetate : Formic acid : Glacialacetic acid : Water

100 11 11 26

Sample preparation
: 1 g of Salacia reticulata crude drug powder was weighed in 250 ml
beaker, extracted with 25 ml of 10% v/v methanolic Demethyl
formamide 4 times and concentrated, cooled and made up and
warmed on steel water bath for 10 minutes. Cooled and made up to
100 ml. 10 µl applied on TLC plate.

Standard preparation : 10 mg of mangiferin was dissolved in 50 ml of 10% methanolic

Demethyl formamide and warmed on steel water bath for 10
minutes. Cooled and made up to 100 ml. 10 µl applied on TLC
plate.

Solvent front run upto : 9 cms

Detection : By spraying 10% H2SO4 in methanol (Fig. 6)

1 2 3 4 5
Fig. 6

1 - Mangiferin
2&3 - RM standard
4&5 - Sample RM

NATURAL REMEDIES-RESEARCH CENTRE 6 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

ANALYTICAL SPECIFICATION FOR SALACIA RETICULATA EXTRACT

12 7(676 /,0,76 35272&2/

1. 'HVFULSWLRQ Light brown to Dark Brown powder.

 3 K \ V L F R F K H P L F D O D Q D O \ V L V

Loss on drying (%w/w) < 5.0

As per USP 24<561> Vegetable Drugs
Ash content (%w/w) < 20.0
Pg.1885
Acid insoluble Ash (%w/w) < 3.0
Total soluble solids (%w/w) > 90.0
pH of 5% w/v solution. 4.5 – 7.5 As per USP<791> Pg.1977
3. 3DUWLFOH 6L]H
As per USP 24 <616> 1913-1914
Bulk Density (g/cc) 0.2 – 0.6
Method – I
Tapped bulk density 0.2 – 0.8
Material passing through 30# > 99.0 As per USP 24 <786> Particle Size
BS/35 ASTM (%w/w) distribution.
4. +HDY\ PHWDO DQDO\VLV

Lead < 10 ppm

AAS / ICP –ES
Cadmium < 1 ppm
Arsenic < 2ppm
5. 0LFURE LRORJLFDO DQDO\VLV

As per FIP Guidelines

Total Viable Aerobic Count < 104 cfu g-1
Total Enterobacteriaceae < 102 org g-1
Total Fungal Count < 102 fs g-1 As per WHO/PHARMA/92.559/Rev.1
6. 7HVW IRU 6SHFLILF 3DWKRJHQ Pg.49-52
As per FIP Guidelines
E.coli (1g) Absent
Salmonella Sp. (10g) Absent
S.aureus (1g) Absent
7. 0\FRWR [LQ DQD O\VLV As per USP 24 Test for Aflatoxins
Aflatoxins (B1 + B2 + G1 + G2) <5ppb Pg.1888
8. 3HVWLFLGH UHVLGXH D QD O\V LV

As per USP & BP Limits

To comply with
Organochlorine Pesticides As per AOAC / USP 24.
USP
Organophosphorus Pesticides
Synthetic pyrethroids
9. 3 K \ W R F K HP L F D O D Q D O \ V L V

Mangiferin (%w/w) Positive By HPLC

10. % L R $ V V D \ V

Alpha Glucosidase Inhibition IC50 (mcg/ml) < 50.0

N o te :

1. Residual pesticide analysis is performed only on request.

NATURAL REMEDIES-RESEARCH CENTRE 7 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

IDENTIFICATION OF EXTRACT BY TLC

Sample detail : Salacia reticulata extract

Adsorbent : Precoated silicagel (Al – Sheet)

Solvent system : Ethyl acetate : Formic acid : Glacialacetic acid : Water

100 11 11 26

Sample preparation : 1 g of Salacia reticulata extract was weighed in 250 ml beaker,

extracted with 25 ml of 10% v/v methanolic Demethyl formamide
4 times and concentrated, cooled and made up and warmed on
steel water bath for 10 minutes. Cooled and made up to 100 ml.
10 µl applied on TLC plate.

Standard preparation : 10 mg of mangiferin was dissolved in 50 ml of 10% methanolic

Demethyl formamide and warmed on steel water bath for 10
minutes. Cooled and made up to 100 ml. 10 µl applied on TLC
plate.

Solvent front run upto : 9 cms

Detection : By spraying 10% H2SO4 in methanol (Fig.)

Fig. 7
S T1 T2 T3 T4
S -MANGIFERIN
T1-T4 - SAMPLE

NATURAL REMEDIES-RESEARCH CENTRE 8 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

QUNATITATIVE DETERMINATION OF MANGIFERIN IN SALACIA RETICULATA

BY HPLC METHOD
Chromatographic system:

1) Shimadazu integrated High Performance Liquid Chromatography system LC/2010 comprising

of system controller unit, degassing unit, low pressure gradient unit, 4 solvents pump unit,
mixer, Auto sampler, column oven, UV-Vis detector and class VP Ver. 6.0 work station is used
for analysis.
2) High Performance Liquid Chromatography system equipped with LC10A pump, SPD-M
10AVP Photo Diode Array Detector in combination with Class VP software is used wherever
PDA analysis is carried out.

Solvent A: Dissolve 0.136 gms of Potassium di-hydrogen orthophosphate (KH2PO4) in 500ml

HPLC grade water, add 0.5ml of orthophosphoric acid (H3PO4) and make upto 1000ml.

Solvent B: 100% Acetonitrile

Gradient conditions:
Time (in min.) Buffer conc. Acetonitrile conc.
0.0 95 5
18.0 55 45
25.0 20 80
28.0 20 80
35.0 55 45
40.0 95 5
45.0 Stop

Chromatographic conditions:

Column: ODS (Octadecyl silance) C18 Phenomenex: 

7\SH /XQD  & 6L]H  [ PP
 PLFURQ 31R  *—4252-E0

Detector: SPD-M 10Avp Photo diode Array Detector or Uv-Vis. Detector.

Wave length: 254nm for monitoring. 190 – 360nm for scanning in PDA

Flow rate: 1.5 ml /min

Injection value: O

Standard preparation: 10 mg of mangiferine was weighed into 100ml volumetric flask dissolved
in 15 ml of Demethyl formamide, warmed on steam water bath for 5 minutes, cooled and the
volume was made up to 100ml with water.

Sample preparation: 1.0 gms of Salacia reticulata extract was weighed into 100 ml volumetric
flask dissolved in 20 ml of Demethyl formamide, warmed on steam water bath for 5-10 minutes,
cooled and made up the volume to 100ml with water.

NATURAL REMEDIES-RESEARCH CENTRE 9 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

HPLC CHROMATOGRAM
SALACIA RETICULATA EXTRACT

Fig. 8

MANGIFERIN – REFERENCE STANDARD

Fig. 9

NATURAL REMEDIES-RESEARCH CENTRE 10 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

BIOLOGICAL STANDARDIZATION

1] Introduction: Intestinal α-Glucosidase inhibitors are

reported to be powerful therapeutic agents in carbohydrate
metabolic disorders, especially diabetes mellitus and
obesity. Postprandial hyperglycemia and hyperinsulinemia
are expected to be diminished by inhibition of poly and
oligosacharide digestion in the intestinal-tract 24. Practically,
a few α-Glucosidase inhibitors of microbial origin viz.,
Acarbose25 are clinically used for the treatment of diabetes
mellitus.

2] Principle: α-Glucosidase activity can be measured in-

vitro by determination of the reducing sugar (Glucose)
arising from hydrolysis of sucrose by α-Glucosidase
enzyme, isolated from small intestine of rat.

3] Materials:

3.1) Enzyme: α-Glucosidase is isolated from small

intestine of rat.
3.2) Substrate : Sucrose - Reducing sugar free (Himedia)
3.3) Inhibitor: Acarbose (Bayer India Ltd.) Fig. 10

3.4) Other reagents: Disodium hydrogen phosphate, sodium dihydrogen phosphate, sucrose
(Himedia laboratories, Mumbai).
3.5) Glucose reagent kit: Glucose oxidase method (Human GmbH, Germany).
3.6) Preparation of working solutions:

3.6.1) Phosphate buffer (80mM) pH 7.0.

A] Sodium dihydrogen phosphate (NaH2PO4, 2H2O) - 12.48 g in 1000 ml of deionised water

B] Disodium hydrogen phosphate anhydrous (Na2HPO4) - 11.35 g in 1000 ml of deionised
water.
Mix 39 ml of solution A with 61 ml of solution B and make up to 200 ml with deionised
water.

3.6.2) Preparation of Enzyme26: Rats are sacrificed, intestine is removed and chilled with ice
cold 80 mM phosphate buffer (pH-7.0). The intestine is then cut open; the mucosa is
scraped off with a piece of glass rod and homogenized in homogenizer with four parts
(v/w) of cold 80 mM buffer (pH-7.0). The tube is chilled with crushed ice during
homogenization. Nuclei and large cell debris are removed by centrifugation at 2000 to
4000 rpm for 10 minutes and supernatant is stored at – 200C. [Protein content =
approximately 0.5 g/dl., by Lowry's method].

NATURAL REMEDIES-RESEARCH CENTRE 11 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

3.6.3) Substrate: Dissolve 1.2665g of sucrose in 100 ml of 80 mM

phosphate buffer pH 7.0.

3.6.4) Reference standard (Inhibitor): Dissolve 50 mg of acarbose in 50 ml of phosphate

buffer and dilute appropriately to get concentration of 5 mcg/ml using phosphate buffer
pH 7.0

4] Procedure:

4.1) α- Glucosidase inhibition bio-assay27: To 50 µl of enzyme, add 250 µl of buffer or test

sample and incubate at 370C for 30 minutes. Add 500 µl of sucrose solution and incubate at
370C for 20 minutes, heat on boiling water bath for 2 minutes to arrest the reaction and cool.
Measure glucose concentration by Glucose oxidase method.

4.2) Glucose estimation (Glucose oxidase method): Mix 50 µl of sample with 500 µl of
glucose reagent (Glucose reagent kit) then incubate at room temperature for 10 minutes.
Finally measure the concentration of glucose using semi auto-analyzer which is calibrated by
using standard glucose.

5] Calculation: The percentage inhibition of α-glucosidase is calculated as follows:

Absorbance (control) – Absorbance (test)

% inhibition = X 100
Absorbance (control)

Note:
a) IC50 is calculated using Finney Computer Program or by regression analysis by plotting glucose
concentration Vs drug concentration.
b) IC50 of Reference Standard 0.3 to 0.8 µg /ml.
c) Appropriate solvent and colour corrections should be done (in case of coloured samples & non-aqueous
solutions).

6] Critical Control Points:

6.1) pH of the buffer solution

6.2) Ionic strength of buffer
6.3) Incubation temperature and time
6.4) Concentration of substrate and enzyme
6.5) Storage conditions.

NATURAL REMEDIES-RESEARCH CENTRE 12 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

ANALYTICAL SPECIFICATION
OF MANGIFERIN

(2β-D-glucopyranosyl -1,3,6,7-tetrahydroxy-9H-Xanthen-9-one)

Mangiferin is a xanthone-C-glucoside isolated from different plant sources mainly Mangifera indica
L., Salacia reticulata L.

HO
O OH
CH2OH
O
HO
OH
HO OH
OH O

C19H18O11 Mol. Wt. 422.35

Fig. 11

TESTS RESULTS

Description Pale yellow powder.

Solubility Soluble in alcohol DMSO and sparingly soluble in
water, acetone.

Identification (by Spectroscopy & Chromatography)

UV absorption1 Exhibits maxima at 366, 316, 260 and 242
TLC & HPTLC Gives single spot
HPLC Retention time matches with the reference standard
FTIR Characteristic of the compound
H NMR Characteristic of the compound
13
C NMR Characteristic of the compound
Mass Spectrum Characteristic of the compound
Purity > 98%w/w

References:

1. S. Ghosal, Phytochemistry, 1971; 10 : 2425-2432

2. Takashi Tanaka, Chem. Pharm. Bull., 1984; 32(7) : 2676-2686.

NATURAL REMEDIES-RESEARCH CENTRE 13 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

UV ABSORPTION
SPECTRUM OF MAGIFERIN
Light absorption: UV absorption of 0.002% w/v solution in alcohol in the range of 225 nm to 500 nm.

Mangiferin Sample Mangiferin Standard

Fig. 12 Fig. 13

0.02072 % w/v solution of sample in alcohol 0.02048 % w/v solution of standard in alcohol
File Name : Mangiferin -Sample File Name : Mangiferin -Standard from
Extrasynthese, France
Created : 10:55 10/06/98 Created : 12:21 10/06/98
Data : Original Data : Original
Measuring Mode : Abs. Measuring Mode : Abs.
Scan Speed : Medium Scan Speed : Medium
Slit Width : 1.0 Slit Width : 1.0
Sampling Interval : 0.2 Sampling Interval : 0.2

No. Wavelength (nm.) Abs. Log ε No. Wavelength (nm.) Abs. Log ε
1 366.00 0.592 4.09 1 366.00 0.607 4.09
2 316.60 0.807 4.22 2 317.00 0.832 4.22
3 258.60 1.600 4.51 3 258.80 1.654 4.52
4 241.60 1.283 4.42 4 241.60 1.333 4.43

The log ε exact matches with the standard and to the reported value* 242 (4.44), 260 (4.50), 316 (4.22), 366 (4.1)

OVERLAY SPECTRA OF STANDARD AND SAMPLE

Fig. 14
* Ref: Xanthones of Canscora decussata Schutt. , R. K. Chaudhari et al., Phytochemistry, 1971; 10 : 2425 - 2432

NATURAL REMEDIES-RESEARCH CENTRE 14 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

TLC PROFILE

Sample : Mangiferin (98%)

Adsorbent : Silica gel 60 F254 (Merck Al. Sheets - 1.05554)

Mobile Phase : Ethyl acetate: Formic acid : Acetic acid : Water

100 : 11 : 11 : 27

Standard & Sample : 0.050% w/v methanolic solution of Mangiferin sample and
preparation standard were prepared. 10µl of this solution is applied on
TLC plate.

Solvent front run upto : 8 cms

Scanning : Densitometer 254 nm

S T
Fig. 15 Fig. 16
S- STANDARD
T- SAMPLE

Applicator instrument for TLC and HPTLC: Linomat IV - CAMAG

Densitometer: Shimadzu Flying Spot Densitometer
Model: CS9301PC

NATURAL REMEDIES-RESEARCH CENTRE 15 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

IR SPECTRUM

)LJ 

1
H NMR

)LJ 

MANGIFERIN

NATURAL REMEDIES-RESEARCH CENTRE 16 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

13
C NMR SPECTRUM

Z
)LJ 

MASS SPECTRUM

)LJ 

MANGIFERIN

NATURAL REMEDIES-RESEARCH CENTRE 17 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

HPLC CHROMATOGRAMS
STANDARD SAMPLE

Fig. 21 Fig. 22

PDA CHROMATOGRAM

Fig. 23

3D VIEW

Fig. 24
MANGIFERIN

NATURAL REMEDIES-RESEARCH CENTRE 18 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

References:

Botanical description and Geographical distribution:

1. Saldanha C.J, “Flora of Karnataka”, Pub: Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi,
1996, 2, 92.

2. Ravikumar K, Ved D.K, “Illustrated field guide- 100 Red listed medicinal plants of conservation
concern in southern India”, Pub: Foundation for revitalisation of local health traditions,
Bangalore, 2000, 327-330.

3. Lawrence G.H.M, “Taxonomy of vascular plants”, Pub: Oxford & IBH Publishing Co. Pvt. Ltd.,
New Delhi, 1951, 578-579.

Traditional uses:

4. “Indian Medicinal plants- a compendium of 500 species”, Pub: Orient longman Ltd., Hyderabad,
1996, 5, 47.

5. Husain A, Virmani O.P et al, “Dictionary of Indian Medicinal plants”, Pub: Central Institute of
Medicinal and Aromatic plants, Lucknow, 1992, 400

6. Nadkarni K. M., “The Indian Materia Medica” Pub:Popular prakashan Pvt. Ltd., Bombay, 1993;
1 : 1089.

Pharmacology:

Antidiabetic activity:

7. Serasinghe S., Serasinghe P.,Yamazaki H., Nishiguchi K., et al, “Oral hypoglycemic effect of
Salacia reticulata in the streptozotocin induced diabetic rat”, Phytotherapy Res. 1990,4, 205-
206.

8. Yoshikawa M., Murakami T., Shimada H., Matsuda H., et al, “Salicinol, Potent Antidiabetic
Principle with Unique Thiosugar Sulfonium Sulfate Structure from the Ayurvedic Traditional
Medicine Salacia reticulata in Sri Lanka and India”, Tetrahedron Letters, 1997, 38(48), 8367-
8370.

9. Yoshikawa M., Nishida N., Shimoda H., Takada M., et al, “Polyphenol constituents from
Salacia species: quantitative analysis of mangiferin with alpha-glucosidase and aldose reductase
inhibitory activities”, Yakugaku Zasshi, 2001, 121(5), 371-378.

10. Yoshikawa M., Murakami T., Yashiro K., Matsuda H., “Kotalanol, a potent alpha-glucosidase
inhibitor with thiosugar sulfonium sulfate structure, from antidiabetic ayurvedic medicine
Salacia reticulata”, Chem. Pharm. Bull., 1998, 46(8), 1339-1340.

11. Shimoda H., Kawamori S., Kawahara Y., “Effects of an aqueous extract of Salacia reticulata, a
useful plant in Sri Lanka, on postprandial hyperglycemia in rats and humans”, Nippon Eiyo
Shokuryo Gakkaishi = Journal of the Japanese Society of Nutrition and Food Science, 1998,
51(5), 279-287.

NATURAL REMEDIES-RESEARCH CENTRE 19 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

12. Ichiki H., Miura T., Kubo M., Ishihara E., et al, “New antidiabetic
compounds, mangiferin and its glucoside” Biol. Pharm. Bull., 1998, 21(12),1389-90.

13. Kajimoto O., Kawamori S., Shimoda H., Kawahara Y, et al, “Effect of diet containing Salacia
reticulata on Mild Type 2 Diabetes in Humans - a placebo-controlled, Cross-over Trial” J. Jap.
Soc. Nutr. Food Nutr., 2000,53(5), 199.

14. Miura T., Iwamato N., Kato M., Ichiki H., Kubo M., et al, “The suppressive effect of mangiferin
with exercise on blood lipids in type 2 diabetes” Biol. Pharm. Bull., 2001, 24(9),1091-1092.

Hyperlipidemic activity:

15. Shimoda H., Kawamori S., Kawahara Y., “Preventive effects of an aqueous extract of Salacia
reticulata on hyperlipidemia in rats”, Nippon Eiyo Shokuryo Gakkaishi = Journal of the
Japanese Society of Nutrition and Food Science, 2000, 53(4),149-154

Hepatoprotective and Antioxidant activity:

16. Yoshikawa M., Ninomiya K., Shimoda H., Nishida N., Matsuda H.,”Hepatoprotective and
antioxidative properties of Salacia reticulata: preventive effects of phenolic constituents on
CCl4-induced liver injury in mice” Biol Pharm Bull, 2002, 25(1), 72-6.

Toxicity:
17. Shimoda H, Fujimura T, Makino K, Yoshijima K, et al, “Safety profile of extractive from trunk
of Salacia reticulate (Celastraceae).” Shokuhin Eiseigaku Zasshi = Journal of the Food
Hygienic Society of Japan, 1999, Vol. 40, No. 3, pp.198-205.

Phytochemistry:

18. Yoshikawa M., Nishida N., Shimoda H., Takada M., Kawahara Y., Matsuda, H, “Polyphenol
constituents from Salacia species: quantitative analysis of mangiferin with alpha-glucosidase
and aldose reductase inhibitory activities”, Yakugaku Zasshi, 2001, 121(5), 371-378.

19. Gunatilaka A.A.L., Dhanahbalsingham B., Karunaratne V., Kikuchi T., Tezuka Y., “Studies on
terpenoids and steroids. Part 27.1Structure of a D:A-Friedo-oleanane Triterpenoid from Salacia
reticulate and revision of the structures of Kokoonol and Kokzeylanol series of Triterpenoids”
Tetrahedron,1993, 49, 10397-10404.

20. Tezuka Y., Kikuchi T., Dhanabalasingham B., Karunaratne V., Gunatilaka A.A.L., J. Nat.
Prod.,1993, 3, 273.

21. Tezuka Y., Kikuchi T., Dhanabalasingham B., Karunaratne V., Gunatilaka A.A.L., “Studies on
terpenoids and steroids, 25. Complete 1H- and 13C-NMR spectral assignments of
salaciquinone, a new 7-oxo-quinonemethide dinortriterpenoid”, Journal of Natural Products,
1994, 57(2), 270-276.

22. Kumar V., Wazeer M.I.M., Wijeratne D.B.T., “21α , 26-Dihyroxy-D: A-Friedooleanan-3-one
from Salacia reticulate var.Diandra (Celastraceae)”, Phytochemistry, 1985, 24(9), 2067-2069.

NATURAL REMEDIES-RESEARCH CENTRE 20 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]

 Salacia reticulata

23. Dhanabalasingham B., Karunaratne V., Tezuka Y., Kikuchi T., Gunatilaka
A.A.L., “Biogenetically important quinonemethides and other triterpenoid constituents of
Salacia reticulata”, Phytochemistry, 1996, 42(5), 1377-1385,

Biological standardization:

24. Matsuo T., odaka H., Ikeda H., “Effect of an intestinal disaccharidase inhibitor (AO-128) on
obesity and diabete,” Am. J. Clin. Nutr., 1992, 55(1), 314S - 317S.

25. Krause H.P., Keup U., Puls W., “Inhibition of disaccharidase digestion in rat intestine by the
alpha-Glucosidase inhibitor Acarbose (BAYg 5421),” Digestion, 1982, 23, 232-238.

26. Dahlquist A., “Method for Assay of Intestinal Disaccharidases,” Anal. Biochem., 1964, 18-25.

27. Vogel G.H., Vogel W.H., Drug Discovery and Evaluation, Pharmacological assay, Springer-
Verlag: Germany; 1997, 588-589.

NATURAL REMEDIES-RESEARCH CENTRE 21 QUALITY CONTROL DEPARTMENT [MD/SR/01-03]