VALORIZING OENOTHERA BIENNIS EVENING PRIMROSE SEEDS IN ROMANIA

1
VALORIZING OENOTHERA BIENNIS (EVENING PRIMROSE)
SEEDS IN ROMANIA

• NATIONAL INSTITUTE FOR CHEMICAL - PHARMACEUTICAL
  RD
• BUCHAREST, 112 Calea Vitan SECT. 3
• TEL [phone] FAX [phone]
• E-MAIL iccf_at_ncpri.ro http//www.ncpri.ro

2
Oenothera biennis (Fam. Onagraceae)

• Origin North America
• Biennial plant.
• Naturalized in Britain found all over the world.
  
• Growing by roadsides, railway banks and waste
  places in dry open soils, meadows and old fields.
• Easily cultivated, it prefers acid, neutral and
  basic (alkaline) well-drained soils and requires
  full sun.
• The plants are very tall, often up to 1.5 m in
  height.

3
Oenothera biennis (Fam. Onagraceae)

• The flower spikes grow on auxiliary branches all
  along the stalk. About 6 cm in diameter, bright
  yellow, have four petals, a cross shaped stigma
  and a refluxed calyx. The flowers open in the
  evening and close up during the day and are
  strongly scented with a delicious sweet perfume
  which attracts pollinating moths. The fruit is an
  oblong 2 cm capsule containing many tiny reddish
  seeds. Evening Primrose is in bloom from June to
  September. The entire plant is edible, gather
  edible roots and leaves in spring may be frozen,
  gather flowers, buds and young seed pods in
  bloom, gather entire plant in fall and dry for
  later herb use.

4
Oenothera biennis (Fam. Onagraceae)

• Evening Primrose is edible and medicinal.
• Was cultivated for its nutritious edible roots
• It is increasingly cultivated for the oil
  contained in its seeds, containing the essential
  gamma-linolenic acid, which is known to help
  prevent hardening of the arteries, heart disease,
  eczema, cirrhosis, rheumatoid arthritis,
  menopause, PMS, multiple sclerosis, and
  hypertension. pressure. It has a positive effect
  on sex hormone response including the hormones
  estrogen and testosterone, aids in lowering
  cholesterol levels, and is important in treating
  cirrhosis of the liver. Tests also demonstrate
  that EPO helps relieve pain and inflammation.

5
CROPPING PRACTICE

• BREEDING SEEDS
• SOWING PERIOD EARLY SPRING
• SEED QUOTA 8 Kg/Ha
• SOWING DEPTH 1 - 1.5 Cm
• ROWS INTERVAL 70 Cm
• SEEDS PRODUCTION 785 Kg/Ha

6
Processing

• O. BIENNIS SEEDS OIL
• All operations are carried in the absence of air
  (under inert gas) and light.
• Oil was extracted from seeds with non-polar
  solvent (n-hexane), at normal temperature.
• The extract is further processed by vacuum
  distillation.
• The oil is stabilized by addition of Vitamin E
  (0.1).
• The yield is 1 kg oil from 7 kg vegetable raw
  material

7
Processing

• POLY-PHENOLIC COMPLEXES from SEEDS
• De-fatted seeds (from oil extraction) are
  extracted in polar solvents.
• After concentration, bio-active compounds are
  isolated by selective extraction in
  ethyl-acetate, concentrated and dried.
• The yield is 6.4 g poly-phenolic complexes from
  1 kg de-fatted seeds.

8
Processing

• POLY-PHENOLIC COMPLEXES from LEAVES
• Dry leaves are extracted in polar solvents.
• Chlorophyl is removed by centrifugation and
  extraction with non-polar solvent.
• Bio-active compounds are isolated by selective
  extraction in ethyl-acetate, concentrated and
  dried.
• The yield is 2.3 g poly-phenolic complexes from
  1 kg dry leaves.

9
Oenothera biennis oil (EPO)

• The oil is extracted from seeds solvent
  extraction, press extraction and supercritical
  fluids are used in oil manufacturing.
• Oil obtained by pressing, without use of solvents
  is better quoted and there is a significant trend
  to eliminate the use of solvents in oil
  processing
• Main applications of EPO are as nutritional
  supplements, mainly in diabetes, premenstrual
  syndrome, asthma and hypercholesterolemia

10
Oenothera biennis oil (EPO)

• Physical-chemical characteristics
• Clear, yellow, oily liquid
• Density, 0,9100 - 0,9500
• Refractive index
• nd20 1,450 1,530
• Acidity index, max. 3,00
• Iodination index, min. 100,0
• Saponification index 170,0 225,0
• Peroxide index, max. 3,0
• Unsaponificable substances, max. 5,0

• Fatty acid content
• Palmitic acid, min. 4.5
• Stearic acid, min. 1
• Linoleic acid , min. 63
• g-Linolenic acid, min. 3
• Microbial contamination
• Pathogenic bacteria, absent
• Non- Pathogenic bacteria max. 1000 cfu/g
• Fungi absent

11
Poly-phenolic complexes

• SEEDS
• Yellow - brown powder
• Water and alcohol soluble
• Bioactive components content
• - poly-phenolic acids (expressed as caffeic
  acid) 7.1
• - flavones (expressed as rutoside) 2.4
• - tri-terpenic acids and sterols (expressed as
  ursolic acid) 19.3

LEAVES Yellow - brown powder Water and alcohol
soluble Bioactive components content - poly-pheno
lic acids (expressed as caffeic acid)
2.4 - flavones (expressed as rutoside)
29.7 - tri-terpenic acids and sterols (expressed
as ursolic acid) 32.3
12
Pharmacological properties

• EPO
• Hepato-protective
• Experimental model In vivo, experimental
  intoxication with carbon tetrachloride on Wistar
  rats reference compound Hepabionta Evaluation
  of biochemical markers in blood and liver and
  hystology
• Hypo-lipemic
• Experimental model In vivo, experimental
  intoxication with Tyloxapol on Wistar rats
  reference compound Lypanthyl. Evaluation of
  cholesterolemia and lipemia
• Anti-platelet-aggregating
• Experimental model Ex vivo, adrenalin induced
  platelet aggregation time in blood samples from
  EPO treated animals (Wistar rats)

1.
13
Pharmacological properties

• POLY-PHENOLIC COMPLEXES
• Anti-oxidant
• Experimental model In vitro, evaluation of lipid
  peroxidation in liver homogenates
• Carbon tetrachloride is used to induce
  free-radical formation in liver homogenates free
  radical formation initiates a reaction cascade
  leading to ROS and peroxidation of plasma
  membrane lipids, resulting in cell injuries and
  cell death. Various assays can quantitate such
  effects chemo-luminescence, TBARS, GSH.

1.
14

• - Experimental animal Wistar rats, male,
  180-220 g, 10 animals/group carbon tetrachloride
  intoxication
• - Treatment 21 days
• - Reference product Hepabionta (160 mg/kg)
• - Dose 0.25, 0.5, 1.0 ml/kg EPO
• - Administration route oral
• - Parameters GSH, Alkali phosphatase, MDA

CHART 1 HEPATO-PROTECTIVE ACTIVITY OF EPO
Biochemical parameters of liver
15

• - Experimental animal Wistar rats, male,
  180-220 g, 10 animals/group carbon tetrachloride
  intoxication
• - Treatment 21 days
• - Reference product Hepabionta (160 mg/kg)
• - Dose 0.25, 0.5, 1.0 ml/kg EPO
• - Administration route oral
• - Parameters Glicemia, TGP, TGO, Alkali
  phosphatase, protein

CHART 2 HEPATO-PROTECTIVE ACTIVITY OF EPO Blood
biochemical parameters
16

• - Experimental animal Wistar rats, male,
  180-220 g, 10 animals/group carbon tetrachloride
  intoxication
• - Treatment 21 days
• - Reference product Hepabionta (160 mg/kg)
• - Dose 0.25, 0.5, 1.0 ml/kg EPO
• - Administration route oral
• - Parameters Glicemia, TGP, TGO, Alkali
  phosphatase, protein

CHART 2 HEPATO-PROTECTIVE ACTIVITY OF EPO Blood
biochemical parameters
17

• - Experimental animal Wistar rats, female,
  180-220 g, 10 animals/group
• - Treatment 15 days
• - Experimental and control groups were
  administered daily 150 mg/kg Tyloxapol
• - Reference product Lypanthyl (45 mg/kg)
• - Dose 10 ml/kg EPO
• - Administration route oral
• - Estimated plasma cholesterol and lipids

CHART 3 HYPO-LIPEMIC EFFECT OF O. BIENNIS OIL
18

• - Experimental animal Wistar rats, female,
  180-220 g, 10 animals/group
• - Treatment 15 days
• - Reference product Acetylsalicilic acid (7
  mg/kg)
• - Dose 10 ml/kg EPO
• - Administration route oral
• - Parameter adrenalin induced platelet
  aggregating time

CHART 4 ANTI-PLATELET AGGREGATING ACTION OF EPO
19

• - experimental model TBARS levels in rat
  liver homogenates, in presence of CCl4
• - concentration range of test compounds 20 - 400
  mg/ml
• The poly-phenolic complexes from O. Biennis seeds
  and leaves demonstrated high efficiency as
  protecting agents against lipid peroxidation. The
  maximal protection determined was 72 (seeds) and
  52 (leaves), with EC50 values of 24.57 mg/ml and
  26.16 mg/ml, respectively.

CHART 5 Antioxidant effects of O. biennis
poly-phenolic complexes - O1 - from seeds, O3 -
from leaves
20
Conclusions

• Oenothera biennis oil has a significant
  hepato-protective activity
• both biochemical markers and hystological studies
  provided support for an efficient protection
  against hepato-toxic agents (CCl4 discussed
  above, but also for allyl-alcohol and).
• Since both cited toxicants are acting by
  free-radical and ROS formation, a certain
  antioxidant potential is also to be expected from
  EPO oil, and on-going tests are dedicated to this
  aspect.
• Since this mechanism is closely related with
  xenobiotics metabolism in liver and with
  increased risks of liver injury with age,
  exposure to chemicals, pollutants, drugs - EPO
  represents a natural factor that may provide good
  protection.
• The tests confirmed the important hypo-lipemic
  and hypo-cholesterolemic action
• The significant action of the poly-phenolic
  complexes represents an additional therapeutic
  tool for protection against oxidative stress,
  both in liver and other tissue. Their potential
  opens a new possibility for advanced uses of this
  plant in pharmaceutical applications.

21
The team

• National Institute for Chemical Pharmaceutical
  RD, BUCHAREST, ROMANIA
• Gabriela Pintilie
• Mihaela Albulescu
• Radu Albulescu
• Misu Moscovici
• Lucia Parvu
• Ileana Paraschiv
• Nicoleta Alexandru
• Doina Dobrovolski
• Svetlana Colceru

Research Center for Medicinal and Aromatic
Plants, Fundulea, Romania R. Stoianov
University of Medicine and Pharmacy, Constanta,
Romania Dragomir Coprean