04/21/2304/21/23

Detection and Quantitation of Major Detection and Quantitation of Major Constituents of a Newly Marketed Constituents of a Newly Marketed

Antidiabetic Herbal Product Antidiabetic Herbal Product

Ladislav Novotny*, Mohammed Abdel-Hamid, Ladislav Novotny*, Mohammed Abdel-Hamid,

Fadia MahmoudFadia Mahmoud

Health Sciences Center, Kuwait University, Kuwait

Diabetes mellitus is a metabolic disease which is characterized by high blood sugar levels over a prolonged period. Symptoms of diabetes include: frequent urination, increased thirst and hunger. If left untreated, diabetes can cause many complications.

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Diabetes mellitus (DM) Diabetes mellitus (DM)

DM is characterized by either insufficient insulin secretion by pancreas or improper body response to insulin.

Types of diabetes: •Type 1 DM results from pancreas' failure to produce enough insulin.•Type 2 DM results from insulin resistance•Gestational DM appears in pregnant women with a history of DM

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Diabetes mellitus Diabetes mellitus

Healthy Life Style and Appropriate

Level of Physical Activity / Fitness.

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Diabetes mellitus - prevention Diabetes mellitus - prevention

• Acute complications: Diabetic ketoacidosis and nonketonic hyperosmolar coma.

• Long-term complications: Cardiovascular diseases, stroke, chronic kidney failure, foot ulcers, neuropathies and optic nerves damage.

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Diabetes mellitus - Complications Diabetes mellitus - Complications

Figures about diabetes:

• In 2014, an estimate of 387 millions of people have DM.

• Type 2 DM represents about 90% of diabetic cases, 8.3% of them are adult population.

• From 2012 to 2014, diabetes results in 1.5 to 4.9 million deaths each year.

• Diabetes increases 2-folds person's risk of death.

• The number of diabetic patients is expected to increase to 592 million by 2035.

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Because of the general awareness of DM, populations tend to use various preparations of natural origin to prevent or treat diabetes. Natural Diabetea is an example of such products – it is a herbal tea product of mixture of plants, which is used for several medical purposes including diabetes.

Natural Diabetea is produced by local company in Philippines and is distributed and marketed by some Asian countries.

• To identify the major active constituents of Natural Diabetea by gas chromatography-mass spectrometry (GC-MS).

• To quantify the identified constituents in Natural Diabetea and individual herbal constituents using standard solutions of reference active compounds.

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Aims Aims of this work:of this work:

• Commercial samples of the tea product “Natural Diabetea” were used.

• Scientific literature (PubMed) was searched for the individual plant constituents of tea.

• The tea mixture and separate individual plant samples were analyzed using GC-MS for the identification and quantification of the active constituents.

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Material and MethodsMaterial and Methods

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Herbal name Latin name

English name %

1 Foetid Cassia Cassia tora L. Sickle Senna / 5

Coffeeweed /

2 Attikka Ficus racemosa L. Cluster Fig Tree 2

3 Madan Syzygium cumini (L.) Skeels Java plum / 2

4

Kubuk Terminalia arjuna Wight & Arn. Arjun Tree 2

5

Chebulic Myrobalan Terminalia chebula Retz. Yellow- or Chebulic 2

Myrobalan

6 Beleric Myrobalan Terminalia bellerica (Gaertn.) Beleric or Bastard 2

Myrobalan

7 Indian gooseberry Phyllanthus emblica L. Indian Gooseberry / 5

8 Nerenchi Tribulus terrestris L. Bindii, Bullhead, 2

Devil's Thorn etc.

9 Fenugreek Trigonella foenum-graecum L. Fenugreek 2

10 Wel penala Cardiospermum halicacabum L. Balloon Plant / 1

11 Cinnamon Cinnamomum zeylanicum Blume Cinnamon / 5

True Cinnamon

12 Black Tea Camellia sinensis (L.) Kuntze Tea Plant / Tea Tree / 65

13 Margosa Azadirachta indica A.Juss. Neem / Indian Lilac 5

Indian Lilac

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GC-MS analyses were conducted using Trace

GC Ultra–ISQ (Thermo Scientific, USA). All

operations and data acquisition were

processed by XCalibar software. Library

search was done using the library NIST MS

Search 2.0 of equipment’s software.

GC-MSGC-MS

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GC - MS MethodGC - MS Method

GC

GC separation was achieved using TG-SQC- GC Column:15 m x 0.25 mm x 0.25 µm (Thermo Scientific). Temperature program:

Rate Temperature oC Hold Time min

Initial 50 1.0Ramp 1 30 280 10.0Total run time: 19 min

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GC-MS MethodGC-MS Method

MS program 

MS transfer-line temperature 250 oC

Ion source temp 200 oC

Ionization mode EI

Mass range, m/z 50-450

Extraction method

1 gm of the herbal mixture (Natural Diabetea®) was extracted by sonication with 10 ml of methanol for 60 min at room temperature. The yellow extract was cleaned by filtration using a Millipore filter 0.45 µm and 1 µl aliquot of the extract was injected into GC-MS.

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ProcedureProcedure

Identification of the active constituents in the tea extract

The MS profiles of the chromatographic peaks in GC chromatograms were matched with those in the MS library for identification. The GC-MS profiles of the tea extract were also compared with those of the pure reference compounds (β-sitosterol, β-Amyrin and lupeol) under the same experimental conditions for confirmation.

 

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ProcedureProcedure

Quantification of the active constituents in the tea extract

The peak areas in GC-Chromatograms for the methanol extracts of the tea product and solutions of reference compounds of known concentrations (3 mg/ml) under the same experimental conditions, were used to determine the approximate concentrations of the active constituents in the tea extracts.

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ProcedureProcedure

Quantification of the active constituents in the tea extract

PAAC

CAC = x CREF

PAREF

CAC - concentration of the active constituent in the tea extract;

PAAC - peak area (extract);

PAREF - peak area (reference)

CREF - concentration of reference substance 1717

ProcedureProcedure

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RESULTSRESULTS

Herbal constituents Herbal constituents of Diabetea Tea® :of Diabetea Tea® :

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2020

Latin name* Use - PubMed Constituents (PubMed)     

Ficus racemosa Anti-diuretic; astringent;Tetracyclic triterpenoids: sitosterol, lupeol, stigmasterol;

 Antidiarrheall; healing promotion; leukoanthocyanins; amyrin; glucose

  Antiinflammatory;    Antihyperglycemic  

Terminalia bellerica Antiplasmodial activitySaponines, triterpenoids, carbohydrates, tannins and proteins;

  Insulin-secretion Gallic acid  Antihyperlipidemic  Terminalia chebula Antihyperglycemic Tannins, phenolic compounds  Antiplasmodial  Cinnamomum zeylanicum Antidiabetics Coumarin  Antimicrobial activity Cinnamaldehyde

Camellia sinensisHypoglycemic, Antihyperglycemic Catechins, gallic acid, caffeine etc.

Cassia tora Antitumor  

 Antihypertensive (ACE inhibitor) Fiber soluble

 Glycemic control, insulin secretion  

  Hypolipidemic  Trigonella foenumgraceum Hypoglycemic Diosgenin and other terpenoid substances  Anticancer  

*Plant content provided by the producer.

Plants declared to be in Natural Diabetea

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Plants declared to be in Natural Diabetea 2Latin name* Use - PubMed Constituents (PubMed)

     

Terminalia arjunaEnhances myocardial functions Tannins, triterpenoid saponins (arjunic acid etc.)

 Antiatherogenic; Cardioprotective

flavonoids (arjunone, luteolin), gallic acid, ellagic acid,

  Antihyperlipidemic phytosterols, trace elements  Antihyperglycemic  

Syzygium cumini Antidiabetic: 2-octanone, hexanol, α-copaene, and α-panasinsene  (islet regeneration    and insulin secretion)    Antioxidant  

Azadirachta indica Anti-inflammatory Triterpenoids, azadirone etc.   Chemopreventive   Antioxidant  Tribulus terrestis Aphrodisiac; Antioxidant Saponins, steroidal saponins

  Hepatoprotective  

  Antihypertensive  Ficus bengalensis Antidiabetic; Analgetic Lectins, flavonoids

  Antiinflammatory; Analgesic  Cardiospermum halicacabum Diuretic; Antioxidant isoquinoline alkaloid - berberine

 Antiinflammatory; Antiparasitic  

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Scientific publications registered in PubMed reported various DM-related effects, such as :

• Antihyperglycemic

• Hypoglycemic

• Antihyperlipidemic

• Hypolipidemic

• Glycemic control

• Supporting insulin secretion

• Islet regeneration

• Antidiabetic

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TriterpenoidsTriterpenoids

Triterpenes are assembled from a five-carbon

isoprene unit through the cytosolic

mevalonate pathway to make a thirty-carbon

compounds. Compared to triterpenes, they

contain some functional groups.

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β-Amyrin β-Sitosterol

Lupeol

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Biosynthesis of TriterpenoidsBiosynthesis of Triterpenoids

ββ-Sitosterol-Sitosterol

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Beta-sitosterol is widely distributed in the

plant kingdom and found in vegetable oils,

nuts, avocados.

β-sitosterol is being studied for its potential

to reduce benign prostatic hyperplasia and

blood cholesterol.

LupeolLupeol

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Lupeol is found in a variety of plants, including mango, Acacia etc.

It has a complex pharmacology, displaying antiprotozoal, antimicrobial, antiinflammatory, antitumor and chemopreventive properties.As an anti-inflammatory agent, lupeol functions primarily on the interleukin system (IL4).

ββ-Amyrin-Amyrin

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The data a scarce but, probably, it is

similar in its biological activities to other

triterpenoid.

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RT: 6.37 - 15.64

7 8 9 10 11 12 13 14 15Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100R

ela

tive

Ab

un

da

nce

6.63

7.44

6.87

7.49

7.12 14.71

9.44 12.648.0614.138.12 8.60 11.06 13.049.67

NL:1.21E9TIC MS teaextractconc3

GC-MS chromatogram of the methanol extract of Natural Diabetea

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Retention times (min) and molecular ion masses (m/z) of the major constituents of Natural Diabetea

as indicated by GC-MS analyses Retention time Molecular ion mass Compound (min) (m/z)

6.6 min 194 Caffeine7.4 min 278 Linoleic acid11.0 min 430 Vitamin E12.6 min 414 β-Sitosterol14.1 min 426 β -Amyrin14.7 min 426 Lupeol

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GC-MS spectrum of Nuga Ficus bengalensis extract

RT: 0.00 - 18.67

0 2 4 6 8 10 12 14 16 18Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

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70

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80

85

90

95

100

Re

lative

Ab

un

da

nce

14.67

14.12

13.25

2.71

NL:5.08E7TIC MS Nugaextract

Retention timesLupeol 14.67 minβ-Amyrin 14.12 min

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GC-MS spectrum of Attikka Ficus racemosa extract

RT: 10.37 - 14.53

10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

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70

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95

100

Re

lativ

e A

bu

nd

an

ce

12.66

11.0712.12

10.6810.38

NL:4.64E7TIC MS Attikkaextract

Retention timesβ-Sitosterol 12.66 min

GC/MS Chromatogram of a standard Chromatogram of a standard mixture of

l

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GC-MS Chromatogram of standard mixture of β-sitosterol, lupeol and β-amyrin

RT: 0.00 - 18.69 SM: 11G

0 2 4 6 8 10 12 14 16 18Time (min)

0

5

10

15

20

25

30

35

40

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50

55

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Re

lative

Ab

un

da

nce

13.93

14.45

13.42

12.7412.42

NL:3.04E9TIC MS LAS_Mix1

β-Amyrin

β-Sitosterol

Lupeol

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Quantification of triterpenoids

β-sitosterol, β-Amyrin and lupeol

Quantification was based on measurement of the peak areas of these components in herbal extracts, in comparison to the reference solutions of known concentrations. Using the previous formula, the concentrations of compounds in the tea sample were:

Lupeol 10 mg/g of tea β-Amyrin 3 mg/g of tea β-Sitosterol 7 mg/g of tea

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ConclusionsConclusions

• Scientific data analysis was performed to identify the possible active constituents of Natural Diabetea herbal product.

• Several active constituents of beneficial therapeutic effects were detected in the herbal product using GC-MS.

• The detected triterpenoids (β-amyrin, lupeol and β-sitosterol) were found at considerable amounts in Natural Diabetea which may account for the hypoglycemic effect of tea.

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Thank you !Thank you !